Cancer associated gene ly6k

ABSTRACT

LY6K is identified herein as a potential biomarker useful for the diagnosis of cancer, such as lung and esophageal cancers, as well as for the prognosis of patients with these diseases. As discussed in detail herein, LY6K is specifically over-expressed in most lung and esophageal cancer tissues examined, and is elevated in the sera of a large proportion of patients with these tumors. Accordingly, LY6K may be used in combination with other tumor markers to significantly improve the sensitivity of cancer diagnosis. LY6K may be used in the treatment of ESCC cells, as demonstrated by the fact that small interfering RNAs (siRNAs) of LY6K suppressed growth of the cancer cells. Moreover, the LY6K molecule is also a likely candidate for development of novel therapeutic approaches, such as antibody therapy.

The present application claims the benefit of U.S. ProvisionalApplication No. 60/952,830, filed Jul. 30, 2007, the entire disclosureof which is hereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

The present invention relates to methods for detecting, diagnosing, andproviding a prognosis for cancer or a predisposition therefore,particularly esophageal and lung cancer. More particularly, the presentinvention relates to methods for detecting, diagnosing, and providing aprognosis of esophageal cancer (EC), for example, esophagealsquamous-cell carcinoma (ESCC), and lung cancer, for example non-smallcell lung cancer (NSCLC), as well as methods of treating and preventingesophageal and lung cancer.

BACKGROUND ART

Aerodigestive tract cancer (including carcinomas of lung, esophagus,oral cavity, pharynx, and larynx) accounts for one-third of all cancerdeaths in the United States and is the most common type of cancer insome areas of the world (Berwick M & Schantz S. Cancer Metastasis Rev.1997 September-December;16(3-4):329-47.). Lung cancer is one of the mostcommon malignant tumors in the world, and non-small cell lung cancer(NSCLC) accounts for nearly 80% of those cases (Jemal A, et al. CACancer J Clin. 2006 March-April;56(2):106-30.). Esophageal squamous-cellcarcinoma (ESCC) is one of the most lethal malignancies of the digestivetract, and, at the time of diagnosis, most patients are already at anadvanced stage (Shimada H, et al. Surgery. 2003 May;133(5):486-94.).

Despite modern surgical techniques combined with various adjuvanttreatment modalities, such as radiotherapy and chemotherapy, the overall5-year survival rate of ESCC patients still remains at 40-60% (Tamoto E,et al. Clin Cancer Res. 2004 Jun. 1;10(11):3629-38.), and that of lungcancer patients is only 15% (Parkin DM. Lancet Oncol. 2001September;2(9):533-43., Naruke T, et al. Ann Thorac Surg. 2001June;71(6):1759-64.). Moreover, most survivors experience asubstantially reduced quality of life. Several tumor markers, such asprogastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE),cytokeratin 19-fragment (CYFRA 21-1), squamous-cell carcinoma antigen(SCC), and carcinoembryonic antigen (CEA), have been shown to beelevated in the serum of lung cancer patients (Rastel D, et al. Eur JCancer. 1994;30A(5):601-6.), whereas SCC, CEA, and CYFRA 21-1 have beenfound to be elevated in the serum of advanced ESCC patients (KawaguchiH, et al. Cancer. 2000 Oct. 1;89(7):1413-7.). However, their sensitivityremains at 20-50%, and no tumor marker has been found to be sufficientlyuseful in the detection of lung cancer and ESCC at potentially curativestage. Furthermore, a limited number of practical prognostic biomarkersis presently available for selection of treatment modalities forindividual patients. Therefore, new diagnostic tools and therapeuticstrategies, such as molecular-targeted agents, antibody therapy, andcancer vaccines, are urgently required (Naruke T, et al. Ann ThoracSurg. 2001 June;71(6):1759-64.).

Cancer-testis antigens (CTAs) are proteins that are highly expressed incancer cells, but not in normal cells, with the exception of cells inreproductive tissues such as testis, ovary, and placenta (Boon T & Old LJ. Curr Opin Immunol. 1997 Oct. 1;9(5):681-3., Scanlan M J, et al.Cancer Immun. 2004 Jan. 23;4:1.). Since the cells from these tissues donot express major histocompatibility complex (MHC) class I molecules,CTAs represent promising targets for immunotherapy and have potential asbiomarker for diagnosis of cancer and monitoring of relapse.

Systematic analysis of expression levels of thousands of genes usingcDNA microarray technology provides an effective approach foridentifying molecules involved in pathways of carcinogenesis or thoseassociated with the efficacy of anti-cancer therapy (Kakiuchi S, et al.Mol Cancer Res. 2003 May;1(7):485-99; Kikuchi T, et al. Oncogene. 2003Apr. 10;22(14):2192-205; Kakiuchi S, et al. Hum Mol Genet. 2004 Dec.15;13(24):3029-43. Epub 2004 Oct. 20; Kikuchi T, et al. Int J Oncol.2006 April;28(4):799-805; Taniwaki M, et al. Int J Oncol. 2006September;29(3):567-75; Yamabuki T, et al. Int J Oncol. 2006June;28(6):1375-84.); some of such genes or their gene products havepotential as target molecules for development of novel therapies and/oras cancer biomarkers.

To identify such molecules, particularly for CTAs, genome-wideexpression profile analysis of 101 lung cancer and 19 ESCC patients,coupled with enrichment of tumor cells by laser-capture microdissection,was performed (Kikuchi T, et al. Oncogene. 2003 Apr. 10;22(14):2192-205;Kakiuchi S, et al. Hum Mol Genet. 2004 Dec. 15;13(24):3029-43. Epub 2004Oct. 20; Kikuchi T, et al. Int J Oncol. 2006 April;28(4):799-805;Taniwaki M, et al. Int J Oncol. 2006 September;29(3):567-75; Yamabuki T,et al. Int J Oncol. 2006 June;28(6):1375-84.). The results were thencompared with the expression profile data of 31 normal human tissues (27adult and 4 fetal organs) (Saito-Hisaminato A, et al. DNA Res. 2002 Apr.30;9(2):35-45; Ochi K, et al. J Hum Genet. 2003;48(4):177-82. Epub 2003Feb. 21.).

To verify the biomedical and clinicopathological significance of therespective gene products, a screening system was established using acombination of the tumor-tissue microarray analysis of clinical lung andesophageal cancer materials and RNA interference (RNAi) techniques(Suzuki C, et al. Cancer Res. 2003 Nov. 1;63(21):7038-41; Ishikawa N, etal. Clin Cancer Res. 2004 Dec. 15;10(24):8363-70; Kato T, et al. CancerRes. 2005 Jul. 1;65(13):5638-46; Furukawa C, et al. Cancer Res. 2005Aug. 15;65(16):7102-10; Ishikawa N, et al. Cancer Res. 2005 Oct.15;65(20):9176-84; Suzuki C, et al. Cancer Res. 2005 Dec.15;65(24):11314-25; Ishikawa N, et al. Cancer Sci. 2006August;97(8):737-45; Takahashi K, et al. Cancer Res. 2006 Oct.1;66(19):9408-19; Hayama S, et al. Cancer Res. 2006 Nov.1;66(21):10339-48; Kato T, et al. Clin Cancer Res. 2007 Jan. 15;13(2 Pt1):434-42; Suzuki C, et al. Mol Cancer Ther. 2007 February;6(2):542-51;Yamabuki T, et al. Cancer Res. 2007 Mar. 15;67(6):2517-25.).

Recent acceleration in identification and characterization of novelmolecular targets for cancer therapy has enhanced development of newtypes of anticancer agents, antibodies and vaccines (Kawaguchi H, et al.Cancer. 2000 Oct. 1;89(7):1413-7.). Molecular-targeted drugs areexpected to be highly specific to malignant cells and, due to theirwell-defined mechanisms of action, have minimal adverse effects. As anapproach to such a goal, one promising strategy combines the power ofgenome-wide expression analysis to effectively screen genes that areover-expressed in cancer cells but scarcely expressed in normal organtissues, with high throughput screening of their protein expressionrelated to clinical outcome by means of tissue microarray as well aswith examining loss of function phenotypes by RNAi systems (Suzuki C, etal. Cancer Res. 2003 Nov. 1;63(21):7038-41; Ishikawa N, et al. ClinCancer Res. 2004 Dec. 15;10(24):8363-70; Kato T, et al. Cancer Res. 2005Jul. 1;65(13):5638-46; Furukawa C, et al. Cancer Res. 2005 Aug.15;65(16):7102-10; Ishikawa N, et al. Cancer Res. 2005 Oct.15;65(20):9176-84; Suzuki C, et al. Cancer Res. 2005 Dec.15;65(24):11314-25; Ishikawa N, et al. Cancer Sci. 2006August;97(8):737-45; Takahashi K, et al. Cancer Res. 2006 Oct.1;66(19):9408-19; Hayama S, et al. Cancer Res. 2006 Nov.1;66(21):10339-48; Kato T, et al. Clin Cancer Res. 2007 Jan. 15;13(2 Pt1):434-42; Suzuki C, et al. Mol Cancer Ther. 2007 February;6(2):542-51;Yamabuki T, et al. Cancer Res. 2007 Mar. 15;67(6):2517-25.). Using thiscombination approach, LY6K was herein discovered to be a novel cancertestis antigen (CTA) whose over-expression not only affect the growth ofthe cancer cells but also correlates with an unfavorable prognosticsignificance in NSCLC patients

LY6K was initially identified by several groups (Accession No. AJ001348;AB 105187; SEQ ID NO: 2 encoded by SEQ ID NO: 1) as an unannotatedtranscript. More recent analysis by bioinformatics classified it as amember belonging to the LY6 family having a high homology to the lowmolecular-weight GPI-anchored molecule (de Nooij-van Dalen A G, et al.Int J Cancer. 2003 Mar. 1;103(6):768-74.). Like others in the LY6family, LY6K has 10 cysteine residues in a conserved position andharbors the sequence structure that, in theory, determines GPIanchoring. Members of the LY6 family believed to possess functionsrelated to cell signaling and/or cell adhesion (Bamezai A & Rock K L.Proc Natl Acad Sci U S A. 1995 May 9;92(10):4294-8.), although theprecise role of LY6K in lung carcinogenesis or its physiologicalfunction in normal cells is presently unknown. Since the LY6K gene islocated at chromosome 8q24, a region of allelic gain in more than halfof lung cancers (Balsara B R, et al. Cancer Res. 1997 Jun.1;57(11):2116-20.), its over-expression may result from amplification orchromosomal aberration at this locus.

GPI-anchored proteins are extracellular proteins anchored in the lipidbilayer surface of plasma membrane by GPI (McConville M J & Menon A K.McConville M J & Menon A K. Mol Membr Biol. 2000January-March;17(1):1-16.). There are several known GPI-anchoredproteins that are applicable to diagnosis of human cancer in certainclinical or pre-clinical settings. Human carcinoembryonic antigen (CEA)is presumed to be such a GPI-anchored protein (GOLD P & FREEDMAN S O. JExp Med. 1965 Mar. 1;121:439-62.), and is highly expressed in asignificant proportion of relatively advanced adenocarcinomas,particularly those from the colon, pancreas, breast, and lung(Hammarstrom S. Semin Cancer Biol. 1999 April;9(2):67-81.). Its presencein the serum of cancer patients has been used for disease staging and asan indicator of residual disease and/or tumor recurrences (HammarstromS. Semin Cancer Biol. 1999 April;9(2):67-81.). In addition, sometumor-specific markers and prognostic markers, such as CD109, glypican-3(GPC3), CEA-related cell adhesion molecule 6 (CEACAM6), and prostatestem cell antigen (PSCA) are also categorized as GPI-anchored proteins(Hashimoto M, et al. Oncogene. 2004 Apr. 29;23(20):3716-20; Nakatsura T,et al. Biochem Biophys Res Commun. 2003 Jun. 20;306(1):16-25; JantscheffP, et al. J Clin Oncol. 2003 Oct. 1;21(19):3638-46; Reiter R E, et al.Proc Natl Acad Sci USA. 1998 Feb. 17;95(4):1735-40.). Among them, CD109and GPC3 are also known to be the cancer-testis antigens. In addition,there are several reports of GPI-anchored proteins acting as animmunotherapeutic target for human cancer. The CEA-TRICOM vaccines,designated as “TRICOM” for its inclusion of the three T-cellco-stimulatory molecules B7-1, ICAM-1, and LFA-3, has been shown tosafely generate significant CEA-specific immune responses againstadvanced cancer in phase I clinical trials (Marshall J L, et al. J ClinOncol 2005;23:720-31.). Recently, two independent studies demonstratedthat a passive immunotherapy approach using an anti-PSCA monoclonalantibody inhibited prostate tumor growth and metastasis formation, andfurther prolonged survival times of mice bearing human prostate cancerxenografts (Ross S, et al. Cancer Res 2002;62:2546-53; Saffran D C, etal. Proc Natl Acad Sci U S A 2001;98:2658-63.).

Thus, while elevated expression of LY6K mRNA in human head-and-necksquamous-cell carcinomas and breast cancers has been previouslydescribed (de Nooij-van Dalen A G, et al. Int J Cancer. 2003 Mar.1;103(6):768-74., Lee J W, et al. Oncol Rep. 2006December;16(6):1211-4.), no report to date has clarified thesignificance of the activation of LY6K in human cancer progression andits potential as a therapeutic target and serological/prognosticbiomarker.

SUMMARY OF THE INVENTION

In view of the above, it is an objective of the present invention is toprovide a method for detecting cancer, diagnosing cancer, monitoring acourse of treatment or providing a prognosis for cancer, or determininga predisposition to cancer, more particularly lung cancer (LC, e.g.,NSCLC) and/or esophageal cancer (EC, e.g., ESCC), in a subject. To thatend, by systematically analyzing the expression levels of thousands ofgenes with cDNA microarray technology, it was herein revealed thatlymphocyte antigen 6 complex, locus K (referred to as “LY6K”; also knownas “HSJ001348”, a cDNA for differentially expressed CO16 gene), a memberof the LY6 family, appears to be a novel CTA that is commonlyover-expressed in primary NSCLCs and ESCCs and is essential for thegrowth and/or survival of cancer cells.

Thus, the present invention provides a method for detecting, diagnosing,monitoring the course of treatment, providing a prognosis, ordetermining a predisposition for cancer, particularly lung cancer (LC,e.g., NSCLC) and/or esophageal cancer (EC, e.g., ESCC) in a subject bydetermining the expression level of the LY6K gene in a biological samplefrom a patient, for example, a solid tissue or bodily fluid sample. Anincrease in the expression level of LY6K detected in a test sample ascompared to a normal control level indicates that the subject (fromwhich the test sample was obtained) suffers from or is at risk ofdeveloping LC and/or EC.

The present invention further provides a cancer assay that combines bothLY6K and CEA/CYFRA 21-1 to increase the sensitivity for the patientswith LC or EC without changing the level of false diagnosis found inhealthy volunteers.

The present invention also provides kits for detecting cancer, such aslung cancer or esophageal cancer, such kits including (i) an immunoassayreagent for determining the level of LY6K in a patient derived sample,such as a blood sample; and (ii) a positive control sample for LY6K. Thekits may further include either or both of (iii) an immunoassay reagentfor determining the level of CEA in a patient sample and a positivecontrol sample for CEA, and (iv) an immunoassay reagent for determiningthe level of CYFRA 21-1 in a patient sample and a positive controlsample for CYFRA 21-1.

The present invention further provides methods of identifying agentsthat inhibit the expression or activity of LY6K by contacting a testcell expressing LY6K with a test compound and determining the level ofLY6K expression or the activity of its gene product. The test cell canbe an epithelial cell, for example, an epithelial cell obtained from anesophageal squamous-cell carcinoma. A decrease in the level ofexpression of LY6K or in the level of the activity of its gene productas compared to an expression or activity level measured in the absenceof the agent indicates that the test agent is an inhibitor of LY6K andcan be used to reduce a symptom of cancer, particularly LC or EC.

The present invention also provides a kit that include a detectionreagent which binds to LY6K nucleic acids or polypeptides. Also providedis an array of nucleic acids that binds to LY6K.

Therapeutic methods of the present invention include methods of treatingor preventing cancer, particularly LC and/or EC, in a subject includingthe step of administering to the subject a composition containing one ormore antisense oligonucleotides. In the context of the presentinvention, the antisense composition should be capable of reducing theexpression of LY6K. Accordingly, the antisense composition can containone or more nucleotides which are complementary to LY6K sequences, SEQID NO: 1.

Alternatively, the present methods can include the step of administeringto a subject a composition containing one or more small interfering RNA(siRNA) oligonucleotides. In the context of the present invention, thesiRNA composition should be capable of reducing the expression of LY6Knucleic acids. Examples of siRNA against a Homo sapiens lymphocyteantigen 6 complex, locus K (LY6K) (SEQ ID NO; 1, 2) suitable forinhibiting proliferation and viability of lung cancer cells and/oresophageal cancer cells are described herein. Thus, in some embodimentsof the present invention, LY6K serves a therapeutic target for lungcancer and/or esophageal cancer.

The present invention also provides vaccines and vaccination methods.For example, methods of treating or preventing cancer, for example LCand/or EC, in a subject may involve administering to the subject avaccine composition composed of LY6K polypeptides or immunologicallyactive fragments of such polypeptides. In the context of the presentinvention, an immunologically active fragment is a polypeptide that isshorter in length than the full-length, naturally-occurring protein yetwhich is sufficient to induce an immune response analogous to thatinduced by the full-length protein. For example, an immunologicallyactive fragment is in most cases at least 8 residues in length andcapable of stimulating an immune cell including, a T cell or a B cell.Immune cell stimulation can be measured by detecting cell proliferation,elaboration of cytokines (e.g., IL-2), or production of an antibody.See, for example, Harlow and Lane, Using Antibodies: A LaboratoryManual, 1998, Cold Spring Harbor Laboratory Press; and Coligan, et al.,Current Protocols in Immunology, 1991-2006, John Wiley & Sons.

One advantage of the methods described herein is that cancer,particularly lung cancer and/or esophageal cancer, can be identified ata very early and potentially curative stage, generally prior todetection of overt clinical symptoms.

Regarding the specific aims and objectives recited above, it will beunderstood by those skilled in the art that one or more aspects of thisinvention can meet certain objectives, while one or more other aspectscan meet certain other objectives. Each objective may not apply equally,in all its respects, to every aspect of this invention. As such, theobjects herein can be viewed in the alternative with respect to any oneaspect of this invention. Additional objects and features of theinvention will become more fully apparent when the following detaileddescription is read in conjunction with the accompanying figures andexamples. However, it is to be understood that both the foregoingsummary of the invention and the following detailed description are of apreferred embodiment, and not restrictive of the invention or otheralternate embodiments of the invention. In particular, while theinvention is described herein with reference to a number of specificembodiments, it will be appreciated that the description is illustrativeof the invention and is not constructed as limiting of the invention.Various modifications and applications may occur to those who areskilled in the art, without departing from the spirit and the scope ofthe invention, as described by the appended claims. Likewise, otherobjects, features, benefits and advantages of the present invention willbe apparent from this summary and certain embodiments described below,and will be readily apparent to those skilled in the art. Such objects,features, benefits and advantages will be apparent from the above inconjunction with the accompanying examples, data, figures and allreasonable inferences to be drawn therefrom, alone or with considerationof the references incorporated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments which follows:

FIG. 1. depicts the expression and subcellular localization of LY6K inNSCLCs and ESCCs. Part A depicts the expression of LY6K in 10 clinicalNSCLC samples examined by semiquantitative RT-PCR analysis. Part Bdepicts the expression of LY6K in 8 clinical ESCC samples, as detectedby semi-quantitative RT-PCR analysis. Part C depicts the expression ofLY6K protein in four representative pairs of NSCLC samples (left panels)and four lung-cancer cell lines (right panels), examined by western-blotanalysis. Part D, (left panels) depicts the subcellular localization ofendogenous LY6K protein in lung-cancer cells. LY6K is stained at thecytoplasm of the cell with granular appearance in LC319 and NCI-H1373cells, but not in NCI-H226 and A427 cells(right panels). Measurement ofsecreted LY6K levels with ELISA in culture medium of LY6K-expressingLC319 and NCI-H1373 cells, and non-expressing NCI-H226 and A427 cells.

FIG. 2. depicts the expression of LY6K in normal organ tissues as wellas lung SCC tissues. In Part A, results of Northern blot analysis of theLY6K transcript in 23 normal adult human tissues are depicted. In PartB, results of immunohistochemical evaluation of LY6K protein inrepresentative normal tissues; adult heart, liver, lung, kidney, andtestis, as well as lung SCC tissues, are depicted.

FIG. 3. depicts the association of LY6K over-expression with poorclinical outcomes for NSCLC and ESCC patients. In Part A, results ofimmunohistochemical evaluation of LY6K expression on tumor tissuemicroarrays are depicted (upper panels, X100; lower panels, X200).Examples are shown of strong, weak, and absent LY6K expressions incancer tissues, and of no expression in normal tissues; lung SCC andnormal lung. In Part B, results of Kaplan-Meier analysis of survival ofpatients with NSCLC are depicted (P=0.0026 by the Log-rank test). InPart C, results of immunohistochemical evaluation of LY6K expression ontumor tissue microarrays are depicted (upper panels, X100; lower panels,X200). Examples are shown of strong, weak, and absent LY6K expressionsin cancer tissues, and of no expression in normal tissues; ESCC andnormal esophagus. In Part D, results of Kaplan-Meier analysis ofsurvival of patients with ESCC (P=0.0455 by the Log-rank test) accordingto the expression levels of LY6K are depicted.

FIG. 4. depicts the serologic concentration of LY6K determined by ELISAin serum of patients with lung cancers or esophageal cancers and inhealthy controls or non-neoplastic lung-disease patients with COPD. InPart A, the distribution of LY6K in sera from patients with lung ADC,lung SCC, and ESCC is depicted. Averaged serum levels are shown underthe panel. Differences were significant between lung ADC patients andhealthy individuals (P<0.0001, Mann-Whitney U test), between lung SCCpatients and healthy individuals (P=0.0145) and between ESCC patientsand healthy individuals (P<0.0001). In Part B, the distribution of LY6Kin sera from patients at various clinical stages of lung cancers andesophageal cancers is depicted.

FIG. 5. depicts the serologic concentration of LY6K, CEA and CYFRA 21-1determined by ELISA in serum of patients with lung cancers or esophagealcancers. In Part A, (left panel) ROC curve analysis of LY6K and CEA asserum markers for NSCLC is depicted (X-axis, 1-specificity; Y-axis,sensitivity). The cut-off level was set to provide optimal diagnosticaccuracy and likelihood ratios (minimal false negative and falsepositive results) for LY6K, i.e., 157.0 pg/ml. (right panel)Relationship between serum levels of LY6K and CEA (X-axis, LY6Kconcentration; Y-axis, CEA concentration). In Part B, (left panel) ROCcurve analysis of LY6K and CYFRA 21-1 as serum markers for NSCLC isdepicted (X-axis, 1-specificity; Y-axis, sensitivity). The cut-off levelwas set to provide optimal diagnostic accuracy and likelihood ratios(minimal false negative and false positive results) for LY6K, i.e.,157.0 pg/ml. (right panel) Relationship between serum levels of LY6K andCYFRA 21-1 (X-axis, LY6K concentration; Y-axis, CYFRA 21-1concentration).

FIG. 6. depicts the relationship among three serum markers in NSCLCs.Part A (left panel) depicts the relationship between serum levels of CEAand CYFRA 21-1 for NSCLC patients (X-axis, CEA concentration; Y-axis,CYFRA 21-1 concentration), (middle panel) the relationship between serumlevels of LY6K and CEA (X-axis, LY6K concentration; Y-axis, CEAconcentration), and (right panel) the relationship between serum levelsof LY6K and CYFRA 21-1 (X-axis, LY6K concentration; Y-axis, CYFRA 21-1concentration). In Part B, combinations of CEA, CYFRA 21-1 and LY6K forNSCLC diagnosis are depicted.

FIG. 7. depicts the relationship among three serum markers in ESCCs.Part A (left panel) depicts the relationship between serum levels of CEAand CYFRA 21-1 for ESCC patients (X-axis, CEA concentration; Y-axis,CYFRA 21-1 concentration), (middle panel) the relationship between serumlevels of LY6K and CEA (X-axis, LY6K concentration; Y-axis, CEAconcentration), and (right panel) the relationship between serum levelsof LY6K and CYFRA 21-1 (X-axis, LY6K concentration; Y-axis, CYFRA 21-1concentration). In Part B, combinations of CEA, CYFRA 21-1 and LY6K forESCC diagnosis are depicted.

FIG. 8. depicts the serologic concentration of LY6K determined by ELISAin serum of patients with lung cancers or esophageal cancers. Part Adepicts serologic concentration of LY6K before and after surgery(postoperative days at 2 months) in patients with NSCLC and ESCC. Adotted line indicates the cut-off level for LY6K (157.0 pg/ml). Part Bdepicts serum LY6K levels (pg/ml) and the expression levels of LY6K inprimary tumor tissues in the same NSCLC patients. ‘Score’ for tumortissue indicates the intensity of LY6K staining that was evaluated usingthe criteria described in Materials and Methods.

FIG. 9. depicts the growth inhibition of NSCLC cells by siRNA againstLY6K. Response of RERF-LC-AI cells or TE8 cells to si-LY6K-1 and -2, orcontrol siRNAs (EGFP or SCR). In Part A, the level of LY6K proteinexpression detected by western-blot analysis in RERF-LC-AI cells treatedwith either control or si-LY6Ks is depicted. In Part B, colony-formationassays using RERF-LC-AI cells transfected with si-LY6K-1 and -2,-EGFP,or -SCR are shown. In Part C, the effect of siRNA against LY6K on cellviability, detected by MTT assays, is depicted. All assays wereperformed three times, and in triplicate wells. In Part D, the level ofLY6K protein expression detected by western-blot analysis in TE8 cellstreated with either control or si-LY6Ks is depicted. In Part E, theeffect of siRNA against LY6K on cell viability, detected by MTT assays,is depicted. All assays were performed three times, and in triplicatewells.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present invention is not limited to thespecific methodologies and protocols herein described, as these may varyin accordance with routine experimentation and optimization. It is alsoto be understood that the terminology used in the description is for thepurpose of describing the particular versions or embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims. It must be noted that as usedherein and in the appended claims, the singular forms “a”, “an”, and“the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “cell” is a reference toone or more cells and equivalents thereof known to those skilled in theart, and so forth.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. However, in case of conflict,the present specification, including definitions, will control.Accordingly, in the context of the present invention, the followingdefinitions apply:

In the context of the present invention, the phrase “control level”refers to an mRNA or protein expression level detected in a controlsample and may include any of (a) a normal control level, (b) a lungcancer specific control level and (c) an esophageal cancer specificcontrol level. A control level can be a single expression pattern from asingle reference population or composed from a plurality of expressionpatterns. For example, in the context of the present invention, thecontrol level can be a database of expression patterns from previouslytested cells. The phrase “normal control level” refers to a level ofgene expression detected in a normal, healthy individual or in apopulation of individuals known not to be suffering from cancer, such aslung cancer or esophageal cancer. A normal individual is one with noclinical symptoms of cancer, either lung cancer or esophageal cancer. Onthe other hand, an “LC control level” or “EC control level” refers to alevel of gene expression found in a population suffering from lungcancer or esophageal cancer, respectively.

Alternatively, a similarity in LY6K expression levels between a testsample and an the LC or EC control indicates that the subject (fromwhich the test sample was obtained) suffers from or is at risk ofdeveloping LC or EC, respectively.

According to the present invention, an expression level of a particulargene is deemed “increased” when expression of the gene is increased byat least 0.1, at least 0.2, at least 1, at least 2, at least 5, or atleast 10 or more fold as compared to a control level. LY6K geneexpression can be determined by detecting mRNA of LY6K from a tissuesample from a patient, e.g., by RT-PCR or Northern blot analysis, ordetecting a protein encoded by LY6K, e.g., by immunohistochemicalanalysis of a tissue sample from a patient.

In the context of the present invention, the tissue sample from apatient may be any tissue obtained from a test subject, e.g., a patientknown to or suspected of having cancer, more particularly LC or EC. Forexample, the tissue can contain epithelial cells. More particularly, thetissue can be epithelial cells from non-small cell lung carcinoma oresophageal squamous-cell carcinoma.

Additional definitions are interspersed in the subsequent text, whereapplicable.

Overview

To identify novel biomarkers and therapeutic targets for cancer,particularly lung and esophageal cancers, genes that were highlytransactivated in a large proportion of non-small cell lung carcinomas(NSCLCs) and esophageal squamous-cell carcinomas (ESCCs) were screenedusing a cDNA microarray representing 27,648 genes. A member of lowmolecular weight, GPI-anchored molecule-like protein, lymphocyte antigen6 complex, locus K (LY6K) was selected as a candidate. Tumor-tissuemicroarray was applied to examine expression of LY6K protein in archivalcancer samples from 413 NSCLC and 271 ESCC patients. Serum LY6K levelsof 112 lung-cancer patients, 81 ESCC patients, and 74 healthy controlswere measured by ELISA. The role of LY6K in cancer cell growth and/orsurvival was then examined by small interfering RNA (siRNA) experiments.

LY6K is abundantly expressed in the great majority of lung andesophageal cancers, while its expression is detected only in testisamong normal tissues. A high level of LY6K expression is also associatedwith poor prognosis of patients with NSCLC (P=0.0026) as well as ESCC(P=0.0455), and multivariate analysis confirms its independentprognostic value for NSCLC (P=0.0201). In fact, the proportion of theserum LY6K-positive cases was 33.9% of NSCLC and 32.1% of ESCC, whileonly 4.1% of healthy volunteers were falsely diagnosed as positive.Furthermore, a combined assay, using both LY6K and carcinoembryonicantigen (CEA), judged 64.7% of the lung adenocarcinoma patients aspositive while 9.5% of healthy volunteers were falsely diagnosed.

CEA is a glycoprotein involved in cell adhesion. It is normally producedduring fetal development; however, the production of CEA stops beforebirth. Therefore, it is not usually found in the blood of healthyadults, although levels are raised in heavy smokers. Furthermore, serumfrom individuals with colorectal, gastric, pancreatic, lung and breastcarcinomas have been shown to possess higher levels of CEA than healthyindividuals. However, CEA results cannot be interpreted as absoluteevidence confirming the presence or absence of malignant disease, butmust be used in conjunction with information from other test proceduresand from clinical evaluations of the patient tested. While CEA levelsare elevated in smokers; patients with inflammation includinginfections, inflammatory bowel disease, and pancreatitis; some patientswith hypothyroidism; cirrhosis; and in some patients with noncolorectalneoplasms especially gastric, pancreatic, breast, and ovarian, it cannotbe considered a suitable screening test for occult cancer. Manynegatives occur in patients with early carcinoma, and even in somepatients with metastatic colorectal and other neoplasms. Therefore,markers that would improve the sensitivity of the assay, particularly inthe context of diagnosing esophageal cancer are in great demand. Asdisclosed herein, LY6K is an example of such a sensitivity improvingmarker.

As demonstrated herein, the use of both LY6K and cytokeratin 19-fragment(CYFRA 21-1) increased assay sensitivity in the detection lungsquamous-cell carcinomas up to 70.4%, while false positive rate wereonly 6.8%. CYFRA 21-1 measures soluble cytokeratin-19 fragments in serumand is a useful marker for lung carcinoma, especially squamous cellcarcinoma. In addition, treatment of NSCLC cells with siRNAs againstLY6K knocked-down its expression and resulted in growth suppression ofthe cancer cells. This data suggests that a cancer-testis antigen LY6Kshould be useful as a diagnostic/prognostic biomarker and probably as atherapeutic target for lung and esophageal cancers.

In sum, the present invention demonstrates that lymphocyte antigen 6complex, locus K (LY6K) (Accession No. AJ001348; AB105187; SEQ ID NO: 2encoded by SEQ ID NO: 1) is a cancer-testis antigen having potential asa biomarker for diagnosis of cancers such as lung and esophageal cancersas well as for assessing and monitoring patients with these diseases.Since serum levels of LY6K are shown herein to elevated in the sera of alarge proportion of the patients, LY6K, combined with other tumormarkers, can significantly improve the sensitivity of cancer diagnosis.It may also find utility as an initial diagnostic for identifyingpatients who might benefit from early systemic treatment. Moreover,LY6K, as an essential contributor to a growth-promoting pathway and toaggressive features of NSCLC and ESCC, is a likely target fordevelopment of therapeutic approaches, such as molecular-targeted drugsand immunotherapies for any types of cancers over-expressing thismolecule.

Diagnosing Lung Cancer and Esophageal Cancer

The expression of the LY6K gene was found to be specifically elevated inpatients with lung cancer or esophageal cancer. Therefore, the geneidentified herein, as well as its transcription and translationproducts, find diagnostic utility as a marker for cancer. Moreparticularly, by measuring the expression of the LY6K gene in a cellsample, lung cancer or esophageal cancer can be diagnosed. Thus, thepresent invention provides a method for diagnosing lung cancer oresophageal cancer or a predisposition for developing lung cancer oresophageal cancer in a subject by determining the expression level ofthe LY6K gene in the subject.

According to the present invention, an intermediate result for examiningthe condition of a subject may be provided. Such intermediate result maybe combined with additional information to assist a doctor, nurse, orother practitioner to determine that a subject suffers from lung canceror esophageal cancer. Alternatively, the present invention may be usedto detect cancerous cells in a subject-derived tissue, and provide adoctor with useful information to determine that the subject suffersfrom lung cancer or esophageal cancer.

The diagnostic method of the present invention involves the step ofdetermining (e.g., measuring) the expression of an LY6K gene. Usingsequence information provided by the GenBank™ database entries for knownsequences, the LY6K gene can be detected and measured using conventionaltechniques well known to one of ordinary skill in the art. For example,sequences within the sequence database entries corresponding to the LY6Kgene can be used to construct probes for detecting RNA sequencescorresponding to the LY6K gene in, e.g., Northern blot hybridizationanalyses. Hybridization probes typically include at least 10, at least20, at least 50, at least 100, or at least 200 consecutive nucleotidesof an LY6K sequence. As another example, the sequences can be used toconstruct primers for specifically amplifying the LY6K nucleic acid in,e.g., amplification-based detection methods, for example,reverse-transcription based polymerase chain reaction. As anotherexample, an antibody against LY6K, e.g., an anti-LY6K polyclonalantibody or anti-LY6K monoclonal antibody, can be used for immunoassay,for example, immunohistochemical analysis, western blot analysis orELISA, etc.

The level of the LY6K gene expression detected in a test cellpopulation, e.g., a tissue sample from a patient, can then be comparedto the expression level(s) of the gene in a reference cell population.The reference cell population may include one or more cells for whichthe compared parameter is known, i.e., non-small lung cancer cells (e.g,LC cells), esophageal squamous-cell carcinoma cells (e.g., EC cells),normal lung epithelial cells (e.g., non-LC cells) or normal esophagealepithelial cells (e.g., non-EC cells).

Whether or not a level of gene expression in a test cell population ascompared to a reference cell population indicates the presence of LC, ECor a predisposition thereto depends upon the composition of thereference cell population. For example, if the reference cell populationis composed of non-LC cells or non-EC cells, a similarity in geneexpression level between the test cell population and the reference cellpopulation indicates the test cell population is non-LC or non-EC.Conversely, if the reference cell population is made up of LC cells orEC cells, a similarity in gene expression between the test cellpopulation and the reference cell population indicates that the testcell population includes LC cells or EC cells.

A level of expression of an LY6K gene in a test cell population isconsidered “altered” or deemed to “differ” if it varies from theexpression level of the LY6K gene in a reference cell population by morethan 1.1, more than 1.5, more than 2.0, more than 5.0, more than 10.0 ormore fold.

Differential gene expression between a test cell population and areference cell population can be normalized to a control nucleic acid,e.g. a housekeeping gene. For example, a control nucleic acid is onewhich is known not to differ depending on the cancerous or non-cancerousstate of the cell. The expression level of a control nucleic acid canthus be used to normalize signal levels in the test and reference cellpopulations. Exemplary control genes include, but are not limited to,e.g., beta actin, glyceraldehyde 3-phosphate dehydrogenase and ribosomalprotein P1.

The test cell population can be compared to multiple reference cellpopulations. Each of the multiple reference cell populations can differin the known parameter. Thus, a test cell population can be compared toa first reference cell population known to contain, e.g., LC cells or ECcells, as well as a second reference cell population known to contain,e.g., non-LC cells or non-EC cells (normal cells). The test cellpopulation can be included in a tissue or cell sample from a subjectknown to contain, or suspected of containing, LC cells or EC cells.

The test cell population can be obtained from a bodily tissue or abodily fluid, e.g., biological fluid (for example, blood, sputum,saliva). For example, the test cell population can be purified from lungtissue or esophageal tissue. Preferably, the test cell populationcomprises an epithelial cell. The epithelial cell is preferably from atissue known to be or suspected to be a non-small cell carcinoma or anesophageal squamous-cell carcinoma.

Cells in the reference cell population are preferably from a tissue typesimilar to that of the test cell population. Optionally, the referencecell population is a cell line, e.g. an LC cell line or an EC cell line(i.e., a positive control) or a normal non-LC cell line or a non-EC cellline (i.e., a negative control). Alternatively, the control cellpopulation can be from a database of molecular information obtained fromcells for which the assayed parameter or condition is known.

The subject is preferably a mammal. Exemplary mammals include, but arenot limited to, e.g., a human, non-human primate, mouse, rat, dog, cat,horse, or cow.

Expression of the LY6K gene disclosed herein can be determined at theprotein or nucleic acid level, using methods known in the art. Forexample, Northern hybridization analysis, using probes whichspecifically recognize one or more of these nucleic acid sequences, canbe used to determine gene expression. Alternatively, gene expression canbe measured using reverse-transcription-based PCR assays, using primersspecific for the LY6K gene sequence e.g., SEQ ID NO: 1 and 2. Expressioncan also be determined at the protein level, i.e., by measuring thelevel of a polypeptide encoded by an LY6K gene, or the biologicalactivity thereof. Such methods are well known in the art and include,but are not limited to, e.g., immunoassays that utilize antibodies toproteins encoded by the genes, e.g., anti-LY6K polyclonal antibodieswhich recognized amino acid sequence comprising SEQ ID NO: 18 or 19described in Example 1, but not limited. The biological activities ofthe proteins encoded by the genes are generally well known and include,e.g., cell proliferative activity. See, Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3^(rd) Edition, 2001, Cold Spring HarborLaboratory Press; Ausubel, Current Protocols in Molecular Biology,1987-2006, John Wiley and Sons; and Harlow and Lane, Using Antibodies: ALaboratory Manual, 1998, Cold Spring Harbor Laboratory Press.

In the context of the present invention, EC or LC may be diagnosed bymeasuring the expression level of LY6K nucleic acids in a testpopulation of cells, (i.e., a biological sample from a patient).Preferably, the test cell population contains an epithelial cell, e.g.,a cell obtained from lung tiassue or esophageal tissue. Gene expressioncan also be measured from blood or other bodily fluids, for example,saliva or sputum. Other biological samples can be used for measuringprotein levels. For example, the protein level in blood or serum from asubject to be diagnosed can be measured by immunoassay or otherconventional biological assay.

Expression of the LY6K gene is first determined in the test cellpopulation or biological sample and then compared to the normal controlexpression level of the LY6K gene. A normal control level corresponds toan expression of the LY6K gene typically found in a cell population froma subject known not to be suffering from LC or EC. An alteration ordifference (e.g., an increase) in the level of expression of the LY6Kgene in a tissue sample from a patient in comparison to expression froma normal control sample indicates that the subject is suffering from oris at risk of developing LC or EC. For example, an increase in theexpression of the LY6K gene in the test cell population as compared tothe expression in a normal control cell population indicates that thesubject is suffering from or is at risk of developing LC or EC.

An increase in expression levels of the LY6K gene in the test cellpopulation as compared to normal control expression levels indicatesthat the subject suffers from or is at risk of developing LC or EC. Forexample, increase in expression levels of at least 1%, at least 5%, atleast 25%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or more of the level of the LY6K gene indicates that thesubject suffers from or is at risk of developing LC or EC.

Screening Assays

Identifying Agents that Inhibit LY6K Gene Expression:

An agent that inhibits the expression of the LY6K gene or the activityof its gene product can be identified by contacting a test cellpopulation that expresses the LY6K gene with a test agent and thendetermining the subsequent level of gene expression or activity of itsgene product. A decrease in the level of gene expression or of activityof its gene product in the presence of the agent as compared to theexpression or activity level in the absence of the test agent indicatesthat the agent is an inhibitor of the LY6K gene and therefore useful ininhibiting LC and EC.

The test cell population can include any cells expressing the LY6K gene.For example, the test cell population can contain epithelial cells, forexample, cells from lung tissue or esophageal tissue. Furthermore, thetest cell population can be an immortalized cell line from a non-smalllung cancer cell or an esophageal squamous-cell carcinoma cell.Alternatively, the test cell population can be composed of cells whichhave been transfected with the LY6K gene or which have been transfectedwith a regulatory sequence (e.g., promoter sequence) from the LY6K geneoperably linked to a reporter gene.

The agent can be, for example, an inhibitory oligonucleotide (e.g., anantisense oligonucleotide, an siRNA or a ribozyme), an antibody, apolypeptide or a small organic molecule. Screening for suitableinhibitory agents can be carried out using high throughput methods, bysimultaneously screening a plurality of agents using multiwell plates(e.g., 96-well, 192-well, 384-well, 768-well, 1536-well). Automatedsystems for high throughput screening are commercially available from,for example, Caliper Life Sciences, Hopkinton, Mass. Small organicmolecule libraries available for screening can be purchased, forexample, from Reaction Biology Corp., Malvern, Pa.; TimTec, Newark, Del.

Identifying Therapeutic Agents:

The differentially expressed LY6K gene disclosed herein can also be usedto identify candidate therapeutic agents for treating LC and EC. Themethods of the present invention therefore involve the screening acandidate therapeutic agent to determine if the test agent can convertan expression level of the LY6K gene that is characteristic of an LCstate or an EC state to a gene expression level characteristic of anon-LC state or a non-EC state.

In the context of the instant method, a test cell population is exposedto a test agent or a plurality of test agents (sequentially or incombination) and the expression of the LY6K gene in the cells ismeasured. The expression level of the gene assayed in the test cellpopulation is compared to the expression level of the same gene in areference cell population that is not exposed to the test agent.

An agent capable of suppressing the expression of the LY6K gene hasmarked clinical benefit. Such agents can be further tested for theability to forestall or prevent lung or esophageal carcinomal growth inanimals or test subjects.

In a further embodiment, the present invention provides methods forscreening candidate agents which act on the targets in the treatment ofLC and/or EC. As discussed in detail above, by controlling theexpression level of the LY6K gene or the activity level of its geneproduct, one can control the onset and progression of LC and/or EC.Thus, candidate agents, which act on the targets in the treatment of LCand/or EC, can be identified through screening methods that use suchexpression and activity levels as indices of the cancerous ornon-cancerous state. In the context of the present invention, suchscreening can include, for example, the following steps:

(a) contacting a test compound with a polypeptide encoded by a LY6Kpolynucleotide

(b) detecting the binding activity between the polypeptide and the testcompound; and

(c) selecting the test compound that binds to the polypeptide.

Alternatively, the screening methods of the present invention caninclude the following steps:

(a) contacting a candidate compound with a cell expressing the LY6Kgene; and

(b) selecting the candidate compound that reduces the expression levelof the LY6K gene, as compared to the expression level detected in theabsence of the candidate compound.

Cells expressing the LY6K gene include, but are not limited to, forexample, cell lines established from LC or EC; such cells can be usedfor the above screening of the present invention.

Alternatively, the screening methods of the present invention caninclude the following steps:

(a) contacting a test compound with a polypeptide encoded by a LY6Kpolynucleotide;

(b) detecting the biological activity of the polypeptide of step (a);and

(c) selecting a compound that suppresses the biological activity of thepolypeptide encoded by the LY6K polynucleotide, as compared to thebiological activity detected in the absence of the test compound.

A protein for use in the screening methods of the present invention canbe obtained as a recombinant protein using the known nucleotide sequencefor the LY6K gene. Based on the information regarding the LY6K gene andits encoded protein, one skilled in the art can select any biologicalactivity of the protein as an index for screening and any suitablemeasurement method to assay for the selected biological activity.Specifically, the LY6K protein is known to have a cell proliferatingactivity. Therefore, the biological activity can be determined usingsuch cell proliferating activity.

Alternatively, the screening methods of the present invention caninclude the following steps:

(a) contacting a candidate compound with a cell into which a vector,containing the transcriptional regulatory region of LY6K genes and areporter gene that is expressed under the control of the transcriptionalregulatory region, has been introduced;

(b) measuring the expression or activity of said reporter gene; and

(c) selecting the candidate compound that reduces the expression oractivity level of said reporter gene, as compared to the expression oractivity level detected in the absence of the candidate compound.

Suitable reporter genes and host cells are well known in the art. Areporter construct suitable for the screening methods of the presentinvention can be prepared by using a transcriptional regulatory regionof the LY6K gene. A nucleotide segment containing the transcriptionalregulatory region can be isolated from a genome library based on thenucleotide sequence information for the LY6K gene.

Selecting a Therapeutic Agent for treating LC and/or EC:

Differences in the genetic makeup of individuals can result indifferences in their relative abilities to metabolize various drugs. Anagent that is metabolized in a subject to act as an anti-LC and/or ECagent can manifest itself by inducing a change in a gene expressionpattern in the subject's cells from that is characteristic of acancerous state to a gene expression pattern that is characteristic of anon-cancerous state. Accordingly, the differentially expressed LY6K geneallows for a putative therapeutic or prophylactic inhibitor of LC and/orEC to be tested in a test cell population from a selected subject inorder to determine if the agent is a suitable inhibitor of LC and/or ECin the subject.

To identify an inhibitor of LC and/or EC that is appropriate for aspecific subject, a test cell population from the subject is exposed toa therapeutic agent, and the expression of the LY6K gene is determined.

In the context of the methods of the present invention, the test cellpopulation contains LC and/or EC cells expressing the LY6K gene.Preferably, the test cell population includes epithelial cells. Forexample, a test cell population can be incubated in the presence of acandidate agent and the pattern of gene expression of the test cellpopulation can be measured and compared to one or more referenceexpression profiles, e.g., an LC reference expression profile, an ECreference expression profile or normal reference expression profile,e.g., a non-LC and non-EC reference expression profile.

A decrease in the expression of the LY6K gene in a test cell populationrelative to a reference cell population containing LC and/or ECindicates that the agent has therapeutic utility. Alternatively, asimilarity in the expression of the LY6K gene in the test cellpopulation and the reference cell population indicates that the agenthas alternate therapeutic utility.

In the context of the present invention, the test agent can be anycompound or composition. Exemplary test agents include, but are notlimited to, immunomodulatory agents (e.g., antibodies), inhibitoryoligonuceotides (e.g., antisense oligonucleodies, short-inhibitoryoligonucleotides and ribozymes) and small organic compounds.

Candidate Compounds:

A compound isolated by the screening assays of the present invention mayserve as a candidate for the development of drugs that inhibit theexpression of the LY6K gene or the activity of the protein encoded bythe LY6K gene and can be applied to the treatment or prevention of lungcancer and/or esophageal cancer.

Moreover, compounds in which a part of the structure of the compoundinhibiting the activity of protein encoded by the LY6K gene is convertedby addition, deletion and/or replacement are also included as thecompounds obtainable by the screening methods of the present invention.

When administrating a compound isolated by the methods of the presentinvention as a pharmaceutical for humans and other mammals, includingwithout limitation, mice, rats, hamsters, guinea-pigs, rabbits, cats,dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, theisolated compound can be directly administered or can be formulated intoa dosage form using known pharmaceutical preparation methods. Forexample, according to the needs of the patient, the drugs can be takenorally, such as in the form of sugar-coated tablets, capsules, elixirsand microcapsules, or non-orally, such as in the form of injections ofsterile solutions or suspensions with water or any otherpharmaceutically acceptable liquid. For example, the compounds can bemixed with pharmaceutically acceptable carriers or media, specifically,sterilized water, physiological saline, plant-oils, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders, and such, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredient contained in such a preparation makes a suitabledosage within the indicated range acquirable.

Examples of additives that can be admixed into tablets and capsulesinclude, but are not limited to, binders, including gelatin, cornstarch, tragacanth gum and arabic gum; excipients, including crystallinecellulose; swelling agents, including corn starch, gelatin and alginicacid; lubricants, including magnesium stearate; sweeteners, includingsucrose, lactose or saccharin; and flavoring agents, includingpeppermint, spearmint, Gaultheria adenothrix oil and cherry. When theunit-dose form is a capsule, a liquid carrier, including an oil, can befurther included in the above ingredients. Sterile composites forinjection can be formulated following normal drug implementations usingvehicles, for example, distilled water or saline solution, suitable forinjection.

Physiological saline, glucose, and other isotonic liquids, includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injection. These can beused in conjunction with suitable solubilizers, for example, alcoholsincluding ethanol; polyalcohols, including propylene glycol andpolyethylene glycol; and non-ionic surfactants, including Polysorbate 80(™) and HCO-50.

Sesame oil or soy-bean oil can be used as an oleaginous liquid, can beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer, and can be formulated with a buffer, including phosphatebuffer and sodium acetate buffer; a pain-killer, including procainehydrochloride; a stabilizer, including benzyl alcohol and phenol; and/oran anti-oxidant. A prepared injection can be filled into a suitableampoule.

Methods well known to those skilled in the art can be used to administerthe pharmaceutical composition of the present invention to patients, forexample as an intraarterial, intravenous, or percutaneous injection oras an intranasal, transbronchial, intramuscular or oral administrationIf said compound is encodable by a DNA, the DNA can be inserted into avector for gene therapy and the vector administered to a patient toperform the therapy. In either context, the dosage and method ofadministration may vary according to the body-weight, age, and symptomsof the patient; however, one skilled in the art can suitably selectthem.

For example, although the dose of a compound that binds to a protein ofthe present invention and regulates its activity depends on thesymptoms, the dose is generally about 0.1 mg to about 100 mg per day,preferably about 1.0 mg to about 50 mg per day and more preferably about1.0 mg to about 20 mg per day, when administered orally to a normaladult human (weighing about 60 kg).

When administering the compound parenterally, in the form of aninjection to a normal adult human (weighing about 60 kg), although thereare some differences according to the patient, target organ, symptomsand method of administration, it is convenient to intravenously inject adose of about 0.01 mg to about 30 mg per day, preferably about 0.1 toabout 20 mg per day and more preferably about 0.1 to about 10 mg perday. In the case of other animals, the appropriate dosage amount can beroutinely calculated by converting to 60 kg of body-weight.

Monitoring and Prognosing Lung Cancer and/or Esophageal Cancer

Assessing the Efficacy of Treatment:

The differentially expressed LY6K gene identified herein also allows forthe course of treatments for LC and/or EC to be monitored and assessed.Alternatively, according to the present invention, an intermediateresult for monitoring the course of treatment of LC and/or EC may beprovided. Such intermediate results may be combined with additionalinformation to assist a doctor, nurse, or other practitioner todetermine that a subject suffers from lung cancer or esophageal cancer.Thus, LY6K gene or protein encoded thereby is useful prognostic markerfor monitoring clinical outcome of LC and/or EC. Alternatively, thepresent invention may be used to detect cancerous cells in asubject-derived tissue, and provide a doctor with useful information toassess the course of treatment of LC and/or EC. In this method, a testcell population is provided from a subject undergoing treatment for LCand/or EC. If desired, test cell populations are obtained from thesubject at various time points, before, during, and/or after treatment.Expression of the LY6K gene in the test cell population is thendetermined and compared to expression of the same genes in a referencecell population which includes cells whose LC state and/or EC state isknown. In the context of the present invention, the reference cellsshould not have been exposed to the treatment of interest.

In the context of monitoring and assessing a particular course oftreatment for LC and/or EC, the biological sample should be derived froma subject undergoing treatment for non-small cell lung cancer and/oresophageal squamous-cell carcinoma. Preferably, multiple test biologicalsamples are obtained from the subject at various time points before,during or after the treatment.

If the reference cell population contains no LC cells and no EC cells, asimilarity in the expression of the LY6K gene in the test cellpopulation and the reference cell population indicates that thetreatment of interest is efficacious. However, a difference in theexpression of the LY6K gene in the test cell population and a normalcontrol reference cell population indicates a less favorable clinicaloutcome or prognosis. Similarly, if the reference cell populationcontains LC cells and/or EC cells, a difference between the expressionof the LY6K gene in the test cell population and the reference cellpopulation indicates that the treatment of interest is efficacious,while a similarity in the expression of the LY6K gene in the testpopulation and an LC control reference cell population and/or an ECcontrol reference cell population indicates a less favorable clinicaloutcome or prognosis.

Additionally, the expression level of the LY6K gene determined in abiological sample from a subject obtained after treatment (i.e.,post-treatment levels) can be compared to the expression level of theLY6K gene determined in a biological sample from a subject obtainedprior to treatment onset (i.e., pre-treatment levels). A decrease in theexpression level in a post-treatment sample indicates that the treatmentof interest is efficacious while an increase or maintenance in theexpression level in the post-treatment sample indicates a less favorableclinical outcome or prognosis.

As used herein, the term “efficacious” indicates that the treatmentleads to a reduction in the expression of LY6K gene or a decrease insize, prevalence, or metastatic potential of LC and/or EC in a subject.When a treatment of interest is applied prophylactically, the term“efficacious” means that the treatment retards or prevents a lung cancerand/or an esophageal tumor from forming or retards, prevents, oralleviates a symptom of clinical LC and/or EC. Assessment of lung oresophageal tumors can be made using standard clinical protocols.

In addition, efficaciousness can be determined in association with anyknown method for diagnosing or treating LC and/or EC. LC and/or EC canbe diagnosed, for example, histopathologically or alternatively byidentifying symptomatic anomalies, e.g., weight loss, loss of appetite,abdominal pain, back pain, anorexia, nausea, vomiting and generalizedmalaise, weakness, and jaundice.

Assessing the Prognosis of a Subject with Lung cancer and/or EsophagealCancer:

The present invention also provides methods for assessing the prognosisof a subject with LC or EC, such methods including the step of comparingthe expression of the LY6K gene in a test cell population to theexpression of the LY6K gene in a reference cell population from patientsover a spectrum of disease stages. By comparing the gene expression ofthe LY6K gene in the test cell population and the reference cellpopulation(s), or by comparing the pattern of gene expression over timein test cell populations from the subject, the prognosis of the subjectcan be assessed.

Alternatively, according to the present invention, an intermediateresult for assessing the prognosis of a subject with LC or EC may beprovided. Such intermediate result may be combined with additionalinformation to assist a doctor, nurse, or other practitioner todetermine that a subject suffers from lung cancer or esophageal cancer.Alternatively, the present invention may be used to detect cancerouscells in a subject-derived tissue, and provide a doctor with usefulinformation to assess the prognosis of a subject with LC or EC.

For example, an increase in the expression of the LY6K gene in a testsample as compared to a normal control sample indicates a less favorableprognosis. Conversely, a similarity in the expression of the LY6K gene,in a test sample as compared to normal control sample, indicates a morefavorable prognosis for the subject.

Treating and Preventing Lung Cancer and/or Esophageal Cancer

Methods of Inhibiting Lung Cancer and/or Esophageal Cancer:

The present invention further provides a method for preventing, treatingand/or alleviating one or more symptoms of LC and/or EC in a subject bydecreasing the expression of the LY6K gene (or the activity of its geneproduct). Suitable therapeutic compounds can be administeredprophylactically or therapeutically to a subject suffering from or atrisk of (or susceptible to) developing LC and/or EC. Prophylacticadministration occurs prior to the manifestation of overt clinicalsymptoms of disease, such that a disease or disorder is prevented oralternatively delayed in its progression. Such subjects can beidentified using standard clinical methods or by detecting an aberrantlevel of expression of the LY6K gene or aberrant activity of its geneproduct. In the context of the present invention, suitable therapeuticagents include, for example, inhibitors of cell cycle regulation, cellproliferation.

The therapeutic methods of the present invention can include the step ofdecreasing the expression, function, or both, of gene product of LY6Kgenes whose expression is aberrantly increased (“up-regulated” or“over-expressed” gene) in lung cells and/or esophageal cells. Expressioncan be inhibited in any of several ways known in the art. For example,expression can be inhibited by administering to the subject a compound,e.g., a nucleic acid that inhibits, or antagonizes the expression of theLY6K gene, e.g., an antisense oligonucleotide or small interfering RNAwhich disrupts expression of the LY6K gene.

Inhibitory Nucleic Acids:

As noted above, inhibitory nucleic acids (e.g., antisenseoligonucleotides, siRNA, ribozymes) complementary to the nucleotidesequence of the LY6K gene can be used to reduce the expression level ofthe gene. For example, inhibitory nucleic acids complementary to theLY6K gene that are up-regulated in lung cancer or esophageal cancer areuseful for the treatment of lung cancer or esophageal cancer.Specifically, the inhibitory nucleic acids of the present invention canact by binding to the LY6K gene, or mRNAs corresponding thereto, therebyinhibiting the transcription or translation of the gene, promoting thedegradation of the mRNA, and/or inhibiting the expression of proteinencoded by the LY6K gene, thereby, inhibiting the function of theprotein.

The term “inhibitory nucleic acids” as used herein encompasses bothnucleotides that are entirely complementary to the target sequence andthose having a mismatch of one or more nucleotides, so long as theinhibitory nucleic acids can specifically hybridize to the targetsequences. The inhibitory nucleic acids of the present invention includepolynucleotides that have a sequence identity of at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or higher over a span of at least 15continuous nucleotides. Algorithms known in the art can be used todetermine the sequence identity.

One useful algorithm is BLAST 2.0, originally described in Altschul etal., (1990) J. Mol. Biol. 215: 403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (available on the World Wide Web atncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89: 10915-9).

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, (1987) J. Mol. Evol. 35: 351-60. The methodused is similar to the method described by Higgins & Sharp, (1989)CABIOS 5:151-3. The program can align, e.g., up to 300 sequences of amaximum length of 5,000 letters. The multiple alignment procedure beginswith the pairwise alignment of the two most similar sequences, producinga cluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendogram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison. For example, in order to determine conserved aminoacids in a monomer domain family or to compare the sequences of monomerdomains in a family, the sequence of the invention, or coding nucleicacids, are aligned to provide structure-function information.

The antisense nucleic acids of the present invention act on cellsproducing the proteins encoded by EC-associated marker genes by bindingto the DNAs or mRNAs encoding the proteins, inhibiting theirtranscription or translation, promoting the degradation of the mRNAs,and inhibiting the expression of the proteins, thereby resulting in theinhibition of the protein function.

An antisense nucleic acid of the present invention can be made into anexternal preparation, for example, a liniment or a poultice, by admixingit with a suitable base material which is inactive against the nucleicacid.

Also, as needed, the antisense nucleic acids of the present inventioncan be formulated into tablets, powders, granules, capsules, liposomecapsules, injections, solutions, nose-drops and freeze-drying agents byadding excipients, isotonic agents, solubilizers, stabilizers,preservatives, pain-killers, and such. These can be prepared byfollowing known methods.

The antisense nucleic acids of the present invention can be given to thepatient by direct application onto the ailing site or by injection intoa blood vessel so that it will reach the site of ailment. Anantisense-mounting medium can also be used to increase durability andmembrane-permeability. Examples include, but are not limited to,liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivativesof these.

The dosage of the inhibitory nucleic acids of the present invention canbe adjusted suitably according to the patient's condition and used indesired amounts. For example, a dose range of 0.1 to 100 mg/kg,preferably 0.1 to 50 mg/kg can be administered.

The antisense nucleic acids of the present invention inhibit theexpression of a protein of the present invention and are thereby usefulfor suppressing the biological activity of the protein of the invention.In addition, expression-inhibitors, including antisense nucleic acids ofthe present invention, are useful in that they can inhibit thebiological activity of a protein of the present invention.

The methods of the present invention can be used to alter LY6Kexpression in a cell. Binding of the antisense nucleic acids to atranscript complementary to the LY6K gene in the target cell results ina reduction in the protein production by the cell. The length of theoligonucleotide is at least 10 nucleotides and can be as long as thenaturally-occurring transcript. Preferably, the oligonucleotide is lessthan 75, 50, 25 nucleotides in length. Most preferably, theoligonucleotide is 19-25 nucleotides in length.

The antisense nucleic acids of present invention include modifiedoligonucleotides. For example, thioated oligonucleotides can be used toconfer nuclease resistance to an oligonucleotide.

The term “polynucleotide” and “oligonucleotide” are used interchangeablyherein unless otherwise specifically indicated and are referred to bytheir commonly accepted single-letter codes. The terms apply to nucleicacid (nucleotide) polymers in which one or more nucleic acids are linkedby ester bonding. The polynucleotide or oligonucleotide may be composedof DNA, RNA or a combination thereof.

As use herein, the term “double-stranded molecule” refers to a nucleicacid molecule that inhibits expression of a target gene including, forexample, short interfering RNA (siRNA; e.g., double-stranded ribonucleicacid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA(siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) orsmall hairpin chimera of DNA and RNA (shD/R-NA)).

Also, an siRNA against the LY6K gene can be used to reduce theexpression level of the LY6K gene. Herein, term “siRNA” refers to adouble stranded RNA molecule which prevents translation of a targetmRNA. Standard techniques for introducing siRNA into the cell can beused, including those in which DNA is a template from which RNA istranscribed. In the context of the present invention, the siRNA iscomposed of a sense nucleic acid sequence and an anti-sense nucleic acidsequence against the LY6K gene. The siRNA is constructed such that asingle transcript has both the sense and complementary antisensesequences from the target gene, e.g., a hairpin. The siRNA may either bea dsRNA or shRNA.

As used herein, the term “dsRNA” refers to a construct of two RNAmolecules having sequences complementary to one another annealedtogether via the complementary sequences to form a double-stranded RNAmolecule. The nucleotide sequence of two strands may include not onlythe “sense” or “antisense” RNAs selected from a protein coding sequenceof target gene sequence, but also RNA molecule having a nucleotidesequence selected from non-coding region of the target gene.

The term “shRNA”, as used herein, refers to an siRNA having a stem-loopstructure, composed of first and second regions complementary to oneanother, i.e., sense and antisense strands. The degree ofcomplementarity and orientation of the regions being sufficient suchthat base pairing occurs between the regions, the first and secondregions being joined by a loop region, the loop resulting from a lack ofbase pairing between nucleotides (or nucleotide analogs) within the loopregion. The loop region of an shRNA is a single-stranded regionintervening between the sense and antisense strands and may also bereferred to as “intervening single-strand”.

As use herein, the term “siD/R-NA” refers to a double-strandedpolynucleotide molecule which is composed of both RNA and DNA, andincludes hybrids and chimeras of RNA and DNA and prevents translation ofa target mRNA. Herein, a hybrid indicates a molecule wherein apolynucleotide composed of DNA and a polynucleotide composed of RNAhybridize to each other to form the double-stranded molecule; whereas achimera indicates that one or both of the strands composing the doublestranded molecule may contain RNA and DNA. Standard techniques ofintroducing siD/R-NA into the cell are used. The siD/R-NA includes aLY6K sense nucleic acid sequence (also referred to as “sense strand”), aLY6K antisense nucleic acid sequence (also referred to as “antisensestrand”) or both. The siD/R-NA may be constructed such that a singletranscript has both the sense and complementary antisense nucleic acidsequences from the target gene, e.g., a hairpin. The siD/R-NA may eitherbe a dsD/R-NA or shD/R-NA.

As used herein, the term “dsD/R-NA” refers to a construct of twomolecules having sequences complementary to one another annealedtogether via the complementary sequences to form a double-strandedpolynucleotide molecule. The nucleotide sequence of two strands mayinclude not only the “sense” or “antisense” polynucleotides sequenceselected from a protein coding sequence of target gene sequence, butalso polynucleotide having a nucleotide sequence selected fromnon-coding region of the target gene. One or both of the two moleculesconstructing the dsD/R-NA are composed of both RNA and DNA (chimericmolecule), or alternatively, one of the molecules is composed of RNA andthe other is composed of DNA (hybrid double-strand).

The term “shD/R-NA”, as used herein, refers to an siD/R-NA having astem-loop structure, including first and second regions complementary toone another, i.e., sense and antisense strands. The degree ofcomplementarity and orientation of the regions being sufficient suchthat base pairing occurs between the regions, the first and secondregions being joined by a loop region, the loop resulting from a lack ofbase pairing between nucleotides (or nucleotide analogs) within the loopregion. The loop region of an shD/R-NA is a single-stranded regionintervening between the sense and antisense strands and may also bereferred to as “intervening single-strand”

The double-stranded molecules of the invention may contain one or moremodified nucleotides and/or non-phosphodiester linkages. Chemicalmodifications well known in the art are capable of increasing stability,availability, and/or cell uptake of the double-stranded molecule. Theskilled person will be aware of other types of chemical modificationwhich may be incorporated into the present molecules (WO03/070744;WO2005/045037). In one embodiment, modifications can be used to provideimproved resistance to degradation or improved uptake. Examples of suchmodifications include phosphorothioate linkages, 2′-O-methyl-4′ linkedribonucleotides, 2′-O-methyl ribonucleotides (especially on the sensestrand of a double-stranded molecule), 2′-deoxy-fluoro ribonucleotides,2′-deoxy ribonucleotides, “universal base” nucleotides, 5′-C-methylnucleotides, and inverted deoxyabasic residue incorporation(US20060122137).

In another embodiment, modifications can be used to enhance thestability or to increase targeting efficiency of the double-strandedmolecule. Modifications include chemical cross linking between the twocomplementary strands of a double-stranded molecule, chemicalmodification of a 3′ or 5′ terminus of a strand of a double-strandedmolecule, sugar modifications, nucleobase modifications and/or backbonemodifications, 2-fluoro modified ribonucleotides and 2′-deoxyribonucleotides (WO2004/029212). In another embodiment, modificationscan be used to increased or decreased affinity for the complementarynucleotides in the target mRNA and/or in the complementarydouble-stranded molecule strand (WO2005/044976). For example, anunmodified pyrimidine nucleotide can be substituted for a 2-thio,5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, anunmodified purine can be substituted with a 7-deza, 7-alkyi, or7-alkenyi purine. In another embodiment, when the double-strandedmolecule is a double-stranded molecule with a 3′ overhang, the3′-terminal nucleotide overhanging nucleotides may be replaced bydeoxyribonucleotides (Elbashir S M et al., Genes Dev 2001 Jan. 15,15(2): 188-200). For further details, published documents such asUS20060234970 are available. The present invention is not limited tothese examples and any known chemical modifications may be employed forthe double-stranded molecules of the present invention so long as theresulting molecule retains the ability to inhibit the expression of thetarget gene.

Furthermore, the double-stranded molecules of the invention may includeboth DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybridpolynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimerapolynucleotide shows increased stability. Mixing of DNA and RNA, i.e., ahybrid type double-stranded molecule consisting of a DNA strand(polynucleotide) and an RNA strand (polynucleotide), a chimera typedouble-stranded molecule including both DNA and RNA on any or both ofthe single strands (polynucleotides), or the like may be formed forenhancing stability of the double-stranded molecule. The hybrid of a DNAstrand and an RNA strand may be the hybrid in which either the sensestrand is DNA and the antisense strand is RNA, or the opposite so longas it has an activity to inhibit expression of the target gene whenintroduced into a cell expressing the gene. Preferably, the sense strandpolynucleotide is DNA and the antisense strand polynucleotide is RNA.Also, the chimera type double-stranded molecule may be either where bothof the sense and antisense strands are composed of DNA and RNA, or whereany one of the sense and antisense strands is composed of DNA and RNA solong as it has an activity to inhibit expression of the target gene whenintroduced into a cell expressing the gene.

In order to enhance stability of the double-stranded molecule, themolecule preferably contains as much DNA as possible, whereas to induceinhibition of the target gene expression, the molecule is required to beRNA within a range to induce sufficient inhibition of the expression. Asa preferred example of the chimera type double-stranded molecule, anupstream partial region (i.e., a region flanking to the target sequenceor complementary sequence thereof within the sense or antisense strands)of the double-stranded molecule is RNA. Preferably, the upstream partialregion indicates the 5′ side (5′-end) of the sense strand and the 3′side (3′-end) of the antisense strand. The upstream partial regionpreferably is a domain consisting of 9 to 13 nucleotides counted fromthe terminus of the target sequence or complementary sequence theretowithin the sense or antisense strands of the double-stranded molecules.Moreover, preferred examples of such chimera type double-strandedmolecules include those having a strand length of 19 to 21 nucleotidesin which at least the upstream half region (5′ side region for the sensestrand and 3′ side region for the antisense strand) of thepolynucleotide is RNA and the other half is DNA. In such a chimera typedouble-stranded molecule, the effect to inhibit expression of the targetgene is much higher when the entire antisense strand is RNA(US20050004064).

In the present invention, the double-stranded molecule may form ahairpin, such as a short hairpin RNA (shRNA) and short hairpinconsisting of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is asequence of RNA or mixture of RNA and DNA making a tight hairpin turnthat can be used to silence gene expression via RNA interference. TheshRNA or shD/R-NA preferably includes the sense target sequence and theantisense target sequence on a single strand wherein the sequences areseparated by a loop sequence. Generally, the hairpin structure iscleaved by the cellular machinery into dsRNA or dsD/R-NA, which is thenbound to the RNA-induced silencing complex (RISC). This complex binds toand cleaves mRNAs which match the target sequence of the dsRNA ordsD/R-NA.

In another embodiment, halogenated RNAs, RNAs partially replaced withDNAs, or methylated RNAs can be used to confer RNAase resistance to thesiRNA. Such nucleic acid derivatives that confer RNAase resistance arealso included in the double-stranded RNA. In the present invention, thedouble stranded molecule may include a double stranded RNA constructedfrom ribonucleotides, modified ribonucleotides, or ribonucleotidederivatives.

An siRNA of the LY6K gene hybridizes to target mRNA and therebydecreases or inhibits production of the polypeptides encoded by the LY6Kgene by associating with the normally single-stranded mRNA transcript,thereby interfering with translation and thus, expression of theprotein. In the context of the present invention, an siRNA is preferablyless than 500, 200, 100, 50, or 25 nucleotides in length. Morepreferably an siRNA is 19-25 nucleotides in length. Exemplary nucleicacid sequence for the production of LY6K siRNA includes the sequences ofnucleotides of SEQ ID NOs: 11 as the target sequence. In order toenhance the inhibition activity of the siRNA, one or more uridine (“u”)nucleotides can be added to 3′end of the antisense strand of the targetsequence. The number of “u's” to be added is at least 2, generally 2 to10, preferably 2 to 5. The added “u's” form a single strand at the 3′endof the antisense strand of the siRNA.

An siRNA of the LY6K gene can be directly introduced into the cells in aform that is capable of binding to the mRNA transcripts. Alternatively,a DNA encoding the siRNA can be carried in a vector.

Vectors can be produced, for example, by cloning an LY6K gene targetsequence into an expression vector having operatively-linked regulatorysequences flanking the sequence in a manner that allows for expression(by transcription of the DNA molecule) of both strands (Lee, N. S., etal., (2002) Nature Biotechnology 20: 500-5). An RNA molecule that isantisense to mRNA of the LY6K gene is transcribed by a first promoter(e.g., a promoter sequence 3′ of the cloned DNA) and an RNA moleculethat is the sense strand for the mRNA of the LY6K gene is transcribed bya second promoter (e.g., a promoter sequence 5′ of the cloned DNA). Thesense and antisense strands hybridize in vivo to generate siRNAconstructs for silencing of the LY6K gene. Alternatively, the twoconstructs can be utilized to create the sense and anti-sense strands ofa siRNA construct. Cloned LY6K gene can encode a construct havingsecondary structure, e.g., hairpins, wherein a single transcript hasboth the sense and complementary antisense sequences from the targetgene.

A loop sequence consisting of an arbitrary nucleotide sequence can belocated between the sense and antisense sequence in order to form thehairpin loop structure. Thus, the present invention also provides siRNAhaving the general formula 5′-[A]-[B]-[A′]-3′,

wherein [A] is a ribonucleotide sequence corresponding to a sequence ofthe LY6K gene,

[B] is a ribonucleotide sequence composed of 3 to 23 nucleotides, and

[A′] is a ribonucleotide sequence having the complementary sequence of[A].

The region [A] hybridizes to [A′], and then a loop composed of region[B] is formed. The loop sequence can be 3 to 23 nucleotides in length.The loop sequence, for example, can be selected from the followingsequences (found on the worldwide web atambion.com/techlib/tb/tb_(—)506.html). Furthermore, a loop sequenceconsisting of 23 nucleotides also provides active siRNA (Jacque, J. M.,et al., (2002) Nature 418 : 435-8.).

CCC, CCACC or CCACACC: Jacque, J. M, et al., (2002) Nature, Vol. 418:435-8.

UUCG: Lee, N. S., et al., (2002) Nature Biotechnology 20: 500-5;Fruscoloni, P., et al., (2003) Proc. Natl. Acad. Sci. USA 100(4):1639-44.

UUCAAGAGA: Dykxhoorn, D. M., et al., (2003) Nature Reviews MolecularCell Biology 4: 457-67.

Accordingly, in some embodiments, the loop sequence can be selected fromgroup consisting of, CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Apreferable loop sequence is UUCAAGAGA (“ttcaagaga” in DNA). Exemplaryhairpin siRNA suitable for use in the context of the present inventioninclude:

for LY6K-siRNA AAGGAGGUGCAAAUGGACAGA-[b]-UCUGUCCAUUUGCACCUCCUU(for target sequence of SEQ ID NO: 11)

The nucleotide sequence of suitable siRNAs can be designed using ansiRNA design computer program available from the Ambion website on theworldwide web at ambion.com/techlib/misc/siRNA_finder.html. The computerprogram selects nucleotide sequences for siRNA synthesis based on thefollowing protocol.

Selection of siRNA Target Sites:

1. Beginning with the AUG start codon of the object transcript, scandownstream for AA dinucleotide sequences. Record the occurrence of eachAA and the 3′ adjacent 19 nucleotides as siRNA target sites. Tuschl, etal. Genes Dev 13(24):3191-7(1999) don't recommend against designingsiRNA to the 5′ and 3′ untranslated regions (UTRs) and regions near thestart codon (within 75 bases) as these may be richer in regulatoryprotein binding sites. UTR-binding proteins and/or translationinitiation complexes can interfere with binding of the siRNAendonuclease complex.

2. Compare the target sites to the human genome database and eliminatefrom consideration any target sequences with significant sequenceidentity to other coding sequences. The sequence identity search can beperformed using BLAST 2.0 (Altschul S F, et al., Nucleic Acids Res.1997;25(17):3389-402; Altschul S F, J Mol Biol. 1990;215(3):403-10.),which can be found on the NCBI server at ncbi.nlm.nih.gov/BLAST/.

3. Select qualifying target sequences for synthesis. Using the Ambionalgorithm, preferably several target sequences can be selected along thelength of the gene to evaluate.

The regulatory sequences flanking the LY6K gene sequences can beidentical or different, such that their expression can be modulatedindependently, or in a temporal or spatial manner. siRNAs aretranscribed intracellularly by cloning the LY6K gene templates,respectively, into a vector containing, e.g., a RNA polymerase IIItranscription unit from the small nuclear RNA (snRNA) U6 or the human H1RNA promoter. For introducing the vector into the cell,transfection-enhancing agent can be used. FuGENE (Roche diagnostics),Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), andNucleofector (Wako pure Chemical) are useful as thetransfection-enhancing agent.

The antisense oligonucleotide or siRNA of the present invention inhibitsthe expression of a polypeptide of the present invention and is therebyuseful for suppressing the biological activity of a polypeptide of theinvention. Also, expression-inhibitors, including the antisenseoligonucleotide or siRNA of the invention, are useful in the point thatthey can inhibit the biological activity of the polypeptide of theinvention. Therefore, a composition composed of one or more antisenseoligonucleotides or siRNAs of the present invention is useful fortreating an esophageal cancer. Alternatively, the present inventionprovides use of inhibitory nucleic acids including antisense nucleicacids or siRNAs, or vector expressing the nucleic acids formanufacturing a pharmaceutical composition for treating or preventing acell proliferative disease, for example cancer, in particular LC and/orEC. Further, the present invention also provides such inhibitory nucleicacids including antisense nucleic acids or siRNAs, or vector expressingthe nucleic acids for treating or preventing a cell proliferativedisease, for example cancer, in particular LC and/or EC.

Antibodies and Immunotherapy:

Alternatively, the function of the LY6K gene products of the genesover-expressed in LC and EC can be inhibited by administering a compoundthat binds to or otherwise inhibits the function of the gene products.For example, the compound can be an antibody which binds to the LY6Kgene product or gene products.

The present invention refers to the use of antibodies, particularlyantibodies against a protein encoded by the LY6K gene, or a fragment ofsuch an antibody. As used herein, the term “antibody” refers to animmunoglobulin molecule having a specific structure, that interacts(i.e., binds) only with the antigen that was used for synthesizing theantibody (i.e., the gene product of an up-regulated marker) or with anantigen closely related thereto. Furthermore, an antibody can be afragment of an antibody or a modified antibody, so long as it binds toone or more of the proteins encoded by the marker genes. For instance,the antibody fragment can be Fab, F(ab′)₂, Fv, or single chain Fv(scFv), in which Fv fragments from H and L chains are ligated by anappropriate linker (Huston J. S. et al. Proc. Natl. Acad. Sci. U.S.A.85:5879-83 (1988)). More specifically, an antibody fragment can begenerated by treating an antibody with an enzyme, including papain orpepsin. Alternatively, a gene encoding the antibody fragment can beconstructed, inserted into an expression vector, and expressed in anappropriate host cell (see, for example, Co M. S. et al. J. Immunol.152:2968-76 (1994); Better M. and Horwitz A. H. Methods Enzymol.178:476-96 (1989); Pluckthun A. and Skerra A. Methods Enzymol.178:497-515 (1989); Lamoyi E. Methods Enzymol. 121:652-63 (1986);Rousseaux J. et al. Methods Enzymol. 121:663-9 (1986); Bird R. E. andWalker B. W. Trends Biotechnol. 9:132-7 (1991)).

An antibody can be modified by conjugation with a variety of molecules,including polyethylene glycol (PEG). The present invention provides suchmodified antibodies. The modified antibody can be obtained by chemicallymodifying an antibody. Such modification methods are conventional in thefield.

An antibody of present invention can be bound to a pharmaceutical agent,wherein the antibody is specific for cancer cells. The pharmaceuticalagent intensively acts on the cancer cells, therefore, even agents withstrong side effects can be used with less side effects, in addition topharmaceutical agents, there are also reports of approaches whereprecursors of pharmaceutical agents, enzymes which metabolize theprecursors to an active form, and so on are bound to the antibodies. Inan alternate embodiment, an antibody of the present invention may befused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to, At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², andradioactive isotopes of Lu.

Alternatively, an antibody can take the form of a chimeric antibodyhaving a variable region from a nonhuman antibody and a constant regionfrom a human antibody, or a humanized antibody, having a complementaritydetermining region (CDR) from a nonhuman antibody, a frame work region(FR) and a constant region from a human antibody. Such antibodies can beprepared by using known technologies. Furthermore, in the presentinvention, an antibody may be a human antibody. For instance, a humanantibody may be selected by screening from phage display library. Methodfor constructing the phage display library and a screening procedure ofsuch antibodies are also well known.

Furthermore, an antibody which has an ADCC or CDC activity and bindsespecially to cancer cells, can be used for treatment of cancer.Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII andFcyRIII. To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).Human effector cells are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcyRI-III and carry out ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred. Complementdependent cytotoxicity (CDC) refer to the ability of a molecule to lysea target in the presence of complement. The complement activationpathway is initiated by the binding of the first component of thecomplement system (Clq) to a molecule (e.g., an antibody) complexed witha cognate antigen. To assess complement activation, a CDC assay, e.g.,as described in Gazzano-Santoro et al. J. Immunol. Methods 202: 163(1996), may be performed.

Other compounds have been developed that target and bind to targets in amanner similar to antibodies. Certain of these “antibody mimics” usenon-immunoglobulin protein scaffolds as alternative protein frameworksfor the variable regions of antibodies. Thus, the term “antibody mimic”refers to non-antibody binding proteins that use non-immunoglobulinprotein scaffolds, including adnectins, avimers, single chainpolypeptide binding molecules, and antibody-like bindingpeptidomimetics, as discussed in more detail below. One of skill willrecognize that any method of using antibodies described in this documentcould also be carried out using antibody mimics.

Ku et al. (Proc. Natl. Acad. Sci. U.S.A. 92(14):6552-6556 (1995))discloses an alternative to antibodies based on cytochrome b562. Ku etal. (1995) generated a library in which two of the loops of cytochromeb562 were randomized and selected for binding against bovine serumalbumin. The individual mutants were found to bind selectively with BSAsimilarly with anti-BSA antibodies.

Lipovsek et al. (U.S. Pat. Nos. 6,818,418 and 7,115,396) discloses anantibody mimic featuring a fibronectin or fibronectin-like proteinscaffold and at least one variable loop. Known as Adnectins, thesefibronectin-based antibody mimics exhibit many of the samecharacteristics of natural or engineered antibodies, including highaffinity and specificity for any targeted ligand. Any technique forevolving new or improved binding proteins can be used with theseantibody mimics.

The structure of these fibronectin-based antibody mimics is similar tothe structure of the variable region of the IgG heavy chain. Therefore,these mimics display antigen binding properties similar in nature andaffinity to those of native antibodies. Further, these fibronectin-basedantibody mimics exhibit certain benefits over antibodies and antibodyfragments. For example, these antibody mimics do not rely on disulfidebonds for native fold stability, and are, therefore, stable underconditions which would normally break down antibodies. In addition,since the structure of these fibronectin-based antibody mimics issimilar to that of the IgG heavy chain, the process for looprandomization and shuffling can be employed in vitro that is similar tothe process of affinity maturation of antibodies in vivo.

Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999))discloses an antibody mimic based on a lipocalin scaffold (Anticalin®).Lipocalins are composed of a beta-barrel with four hypervariable loopsat the terminus of the protein. Beste (1999), subjected the loops torandom mutagenesis and selected for binding with, for example,fluorescein. Three variants exhibited specific binding with fluorescein,with one variant showing binding similar to that of an anti-fluoresceinantibody. Further analysis revealed that all of the randomized positionsare variable, indicating that Anticalin® would be suitable to be used asan alternative to antibodies.

Anticalins® are small, single chain peptides, typically between 160 and180 residues, which provides several advantages over antibodies,including decreased cost of production, increased stability in storageand decreased immunological reaction.

Hamilton et al. (U.S. Pat. No. 5,770,380) discloses a synthetic antibodymimic using the rigid, non-peptide organic scaffold of calixarene,attached with multiple variable peptide loops used as binding sites. Thepeptide loops all project from the same side geometrically from thecalixarene, with respect to each other. Because of this geometricconfirmation, all of the loops are available for binding, increasing thebinding affinity to a ligand. However, in comparison to other antibodymimics, the calixarene-based antibody mimic does not consist exclusivelyof a peptide, and therefore it is less vulnerable to attack by proteaseenzymes. Neither does the scaffold consist purely of a peptide, DNA orRNA, meaning this antibody mimic is relatively stable in extremeenvironmental conditions and has a long life span. Further, since thecalixarene-based antibody mimic is relatively small, it is less likelyto produce an immunogenic response.

Murali et al. (Cell. Mol. Biol. 49(2):209-216 (2003)) discusses amethodology for reducing antibodies into smaller peptidomimetics, theyterm “antibody like binding peptidomemetics” (ABiP) which can also beuseful as an alternative to antibodies.

Silverman et al. (Nat. Biotechnol. (2005), 23: 1556-1561) disclosesfusion proteins that are single-chain polypeptides composed of multipledomains termed “avimers.” Developed from human extracellular receptordomains by in vitro exon shuffling and phage display the avimers are aclass of binding proteins somewhat similar to antibodies in theiraffinities and specificities for various target molecules. The resultingmultidomain proteins can include multiple independent binding domainsthat can exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent App. Pub. Nos. 20040175756,20050048512, 20050053973, 20050089932 and 20050221384.

In addition to non-immunoglobulin protein frameworks, antibodyproperties are also mimicked by compounds composed of RNA molecules andunnatural oligomers (e.g., protease inhibitors, benzodiazepines, purinederivatives and beta-turn mimics) all of which are suitable for use withthe present invention.

Cancer therapies directed at specific molecular alterations that occurin cancer cells have been validated through clinical development andregulatory approval of anticancer drugs, including, for example,trastuzumab (Herceptin) for the treatment of advanced breast cancer,imatinib methylate (Gleevec) for chronic myeloid leukemia, gefitinib(Iressa) for non-small cell lung cancer (NSCLC), and rituximab(anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (CiardielloF and Tortora G. Clin Cancer Res. 2001;7(10):2958-70. Review; Slamon DJ, et al., N Engl J Med. 2001;344(11):783-92; Rehwald U, et al., Blood.2003;101(2):420-4; Fang G, et al., (2000). Blood, 96, 2246-53.). Thesedrugs are clinically effective and better tolerated than traditionalanti-cancer agents because they target only transformed cells. Hence,such drugs not only improve survival and quality of life for cancerpatients, but also validate the concept of molecularly targeted cancertherapy. Furthermore, targeted drugs can enhance the efficacy ofstandard chemotherapy when used in combination with it (Gianni L.(2002). Oncology, 63 Suppl 1, 47-56; Klejman A, et al., (2002).Oncogene, 21, 5868-76.). Therefore, future cancer treatments will likelyinvolve a combination of conventional drugs with target-specific agentsaimed at different characteristics of tumor cells, for example,angiogenesis and invasiveness.

These modulatory methods can be performed ex vivo or in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). The methods involve administeringa protein or combination of proteins or a nucleic acid molecule orcombination of nucleic acid molecules as therapy to counteract aberrantexpression of the differentially expressed genes or aberrant activity oftheir gene products.

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) expression levelsor biological activities of genes and gene products, respectively, canbe treated with therapeutics that antagonize (i.e., reduce or inhibit)activity of the over-expressed gene or genes. Therapeutics thatantagonize activity can be administered therapeutically orprophylactically.

Accordingly, therapeutics that can be utilized in the context of thepresent invention include, e.g., (i) a polypeptide of the LY6K gene, oranalogs, derivatives, fragments or homologs thereof; (ii) antibodies tothe LY6K gene or gene product; (iii) nucleic acids encoding the LY6Kgene; (iv) antisense nucleic acids or nucleic acids that are“dysfunctional” (i.e., due to a heterologous insertion within thenucleic acids of the LY6K gene); (v) small interfering RNA (siRNA); or(vi) modulators (i.e., inhibitors, agonists and antagonists that alterthe interaction between LY6K polypeptide and its binding partner). Thedysfunctional antisense molecules are utilized to “knockout” endogenousfunction of a polypeptide by homologous recombination (see, e.g.,Capecchi, Science 244: 1288-92 1989).

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA of LY6K, by obtaining a patient tissue sample (e.g.,from biopsy tissue) and assaying it in vitro for RNA of LY6K or peptidelevels, structure and/or activity of the LY6K peptides. Methods that arewell known within the art include, but are not limited to, immunoassays(e.g., by Western blot analysis, immunoprecipitation followed by sodiumdodecyl sulfate (SDS) polyacrylamide gel electrophoresis,immunocytochemistry, etc.) and/or hybridization assays to detectexpression of mRNAs (e.g., Northern assays, dot blots, in situhybridization, etc.).

Prophylactic administration occurs prior to the manifestation of overtclinical symptoms of disease or disorder, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.

Therapeutic methods of the present invention can include the step ofcontacting a cell with an agent that modulates one or more of theactivities of the gene products of the LY6K gene. Examples of agentsthat modulates protein activity include, but are not limited to, nucleicacids, proteins, naturally occurring cognate ligands of such proteins,peptides, peptidomimetics, and other small molecule.

Vaccinating Against Lung Cancer and Esophageal Cancer:

The present invention also relates to methods of treating or preventinglung cancer and esophageal cancer in a subject including the step ofadministering to said subject a vaccine containing one or morepolypeptides encoded by LY6K nucleic acid, an immunologically activefragment of said polypeptide (i.e., an epitope), or a polynucleotideencoding such a polypeptide or fragment thereof. Examples of LY6K(URLC10) derived peptide vaccines for treating cancer are described inthe WIPO Publication, WO 2006/90810, the entire contents of which areincorporated by reference herein.

Administration of the polypeptide induces an anti-tumor immunity in asubject. To induce anti-tumor immunity, one or more polypeptides encodedby LY6K nucleic acids, an immunologically active fragment(s) of saidpolypeptides, or polynucleotide(s) encoding such polypeptide(s) orfragment(s) thereof is administered to subject in need thereof.Furthermore, the one or more polypeptides encoded by the LY6K nucleicacids can induce anti-tumor immunity against metastatic and recurrentlung cancer or esophageal cancer, respectively. The polypeptide or theimmunologically active fragments thereof are useful as vaccines againstLC or EC. In some cases, the proteins or fragments thereof can beadministered in a form bound to the T cell receptor (TCR) or presentedby an antigen presenting cell (APC), including macrophages, dendriticcells (DC), or B-cells. Due to the strong antigen presenting ability ofDC, the use of DC is most preferable among the APCs.

Identification of immunologically active fragments (i.e., epitopes) iswell known in the art. B-cell epitopes can be formed both fromcontiguous amino acids or non-contiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding (i.e., conformationally determined)are typically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitopecan be identified in a simple immunoassay showing the ability of oneantibody to block the binding of another antibody to a target antigen(e.g., a competitive ELISA or solid phase radioimmunoassay (SPRIA)).T-cells recognize continuous epitopes of about nine amino acids for CD8cells or about 13-15 amino acids for CD4 cells. T cells that recognizethe epitope can be identified by in vitro assays that measureantigen-dependent proliferation, as determined by ³H-thymidineincorporation by primed T cells in response to an epitope (Burke et al.,J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependent killing(cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. (1996)156:3901-10) or by cytokine secretion. Methods for determiningimmunogenic epitopes are described, for example, in Reineke, et al.,Curr Top Microbiol Immunol (1999) 243:23-36; Mahler, et al., ClinImmunol (2003) 107:65-79; Anthony and Lehmann, Methods (2003) 29:260-9;Parker and Tomer, Methods Mol Biol (2000) 146:185-201; DeLisser, MethodsMol Biol (1999) 96:11-20; Van de Water, et al., Clin ImmunolImmunopathol (1997) 85:229-35; Carter, Methods Mol Biol (1994)36:207-23; and Pettersson, Mol Biol Rep (1992) 16:149-53.

In the present invention, a vaccine against LC and/or EC refers to asubstance that has the ability to induce anti-tumor immunity uponinoculation into animals. According to the present invention,polypeptides encoded by the LY6K gene, or fragments thereof, are HLA-A24or HLA-A*0201 restricted epitopes peptides that induce potent andspecific immune response against LC and/or EC cells expressing the LY6Kgene. Thus, the present invention also encompasses methods of inducinganti-tumor immunity using the polypeptides. In general, anti-tumorimmunity includes immune responses including as follows:

induction of cytotoxic lymphocytes against tumors,

induction of antibodies that recognize tumors, and

induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immuneresponses upon inoculation into an animal, the protein is determined tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a protein can be detected by observing in vivo orin vitro the response of the immune system in the host against theprotein.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. Specifically, a foreign substance that entersthe living body is presented to T cells and B cells by the action ofantigen presenting cells (APCs). T cells that respond to the antigenpresented by the APCs in an antigen specific manner differentiate intocytotoxic T cells (or cytotoxic T lymphocytes; CTLs) due to stimulationby the antigen, and then proliferate (this is referred to as activationof T cells). Therefore, CTL induction by a certain peptide can beevaluated by presenting the peptide to a T cell via an APC, anddetecting the induction of CTLs. Furthermore, APCs have the effect ofactivating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NKcells. Since CD4+ T cells and CD8+ T cells are also important inanti-tumor immunity, the anti-tumor immunity-inducing action of thepeptide can be evaluated using the activation effect of these cells asindicators. See, Coligan, Current Protocols in Immunology, supra.

A method for evaluating the inducing action of CTLs using dendriticcells (DCs) as the APC is well known in the art. DCs are arepresentative APCs having the strongest CTL-inducing action among APCs.In this method, the test polypeptide is initially contacted with DCs,and then the DCs are contacted with T cells. Detection of T cells havingcytotoxic effects against the cells of interest after the contact withDC shows that the test polypeptide has an activity of inducing thecytotoxic T cells. Activity of CTLs against tumors can be detected, forexample, using the lysis of ⁵¹Cr-labeled tumor cells as the indicator.Alternatively, methods of evaluating the degree of tumor cell damageusing ³H-thymidine uptake activity or LDH (lactosedehydrogenase)-release as the indicator is also well known.

Apart from DCs, peripheral blood mononuclear cells (PBMCs) can also beused as the APC. The induction of CTLs has been reported to be enhancedby culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTLshave been shown to be induced by culturing PBMCs in the presence ofkeyhole limpet hemocyanin (KLH) and IL-7.

Test polypeptides confirmed to possess CTL-inducing activity by thesemethods are deemed to be polypeptides having DC activation effect andsubsequent CTL-inducing activity. Therefore, polypeptides that induceCTLs against tumor cells are useful as vaccines against tumors.Furthermore, APCs that have acquired the ability to induce CTLs againsttumors through contact with the polypeptides are also useful as vaccinesagainst tumors. Furthermore, CTLs that have acquired cytotoxicity due topresentation of the polypeptide antigens by APCs can be also be used asvaccines against tumors. Such therapeutic methods for tumors, usinganti-tumor immunity due to APCs and CTLs, are referred to as cellularimmunotherapy.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the

CTL-induction is known to be increased by combining a plurality ofpolypeptides having different structures and contacting them with DCs.Therefore, when stimulating DCs with protein fragments, it isadvantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide canbe confirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide, and when growthof tumor cells is suppressed by those antibodies, the polypeptide isdeemed to have the ability to induce anti-tumor immunity.

Anti-tumor immunity is induced by administering the vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of LC and/or EC. Therapy against cancer or prevention ofthe onset of cancer includes any of the following steps, includinginhibition of the growth of cancerous cells, involution of cancer, andsuppression of the occurrence of cancer. A decrease in mortality andmorbidity of individuals having cancer, decrease in the levels of tumormarkers in the blood, alleviation of detectable symptoms accompanyingcancer, and such are also included in the therapy or prevention ofcancer. Such therapeutic and preventive effects are preferablystatistically significant. For example, in observation, at asignificance level of 5% or less, wherein the therapeutic or preventiveeffect of a vaccine against cell proliferative diseases is compared to acontrol without vaccine administration. For example, Student's t-test,the Mann-Whitney U-test, or ANOVA can be used for statistical analysis.

The above-mentioned protein having immunological activity or a vectorencoding the protein can be combined with an adjuvant. An adjuvantrefers to a compound that enhances the immune response against theprotein when administered together (or successively) with the proteinhaving immunological activity. Exemplary adjuvants include, but are notlimited to, cholera toxin, salmonella toxin, alum, and such, but are notlimited thereto. Furthermore, the vaccine of this invention can becombined appropriately with a pharmaceutically acceptable carrier.Examples of such carriers include sterilized water, physiologicalsaline, phosphate buffer, culture fluid, and such. Furthermore, thevaccine can contain as necessary, stabilizers, suspensions,preservatives, surfactants, and such. The vaccine can be administeredsystemically or locally, for example, through intradermal,intramuscular, subcutaneous, transdermal, buccal, or intranasal routes.Vaccine administration can be performed by single administration, orboosted by multiple administrations. Doses are as set forth below.

When using an APC or CTL as the vaccine of this invention, tumors can betreated or prevented, for example, by the ex vivo method. Morespecifically, PBMCs of the subject receiving treatment or prevention arecollected, the cells are contacted with the polypeptide ex vivo, andfollowing the induction of APCs or CTLs, the cells can be administeredto the subject. APCs can be also induced by introducing a vectorencoding the polypeptide into PBMCs ex vivo. APCs or CTLs induced invitro can be cloned prior to administration. By cloning and growingcells having high activity of damaging target cells, cellularimmunotherapy can be performed more effectively. Furthermore, APCs andCTLs isolated in this manner can be used for cellular immunotherapy notonly against individuals from whom the cells are retrieved, but alsoagainst similar types of tumors from other individuals.

General methods for developing vaccines are described, for example, inVaccine Protocols, Robinson and Cranage, Eds., 2003, Humana Press;Marshall, Vaccine Handbook: A Practical Guide for Clinicians, 2003,Lippincott Williams & Wilkins; and Vaccine Delivery Strategies,Dietrich, et al., Eds., 2003, Springer Verlag.

Pharmaceutical Compositions:

Furthermore, a pharmaceutical composition for treating or preventing acell proliferative disease, for example cancer, in particular LC and/orEC, containing a pharmaceutically effective amount of the polypeptide ofthe present invention is provided. The pharmaceutical composition can beused for raising anti tumor immunity. Alternatively, the presentinvention provides use of LY6K protein or gene encoding the protein formanufacturing a pharmaceutical composition for treating or preventing acell proliferative disease, for example cancer, in particular LC and/orEC. Further, the present invention also provides LY6K protein or geneencoding the protein for treating or preventing a cell proliferativedisease, for example cancer, in particular LC and/or EC.

In the context of the present invention, suitable pharmaceuticalformulations include, but are not limited to, those suitable for oral,rectal, nasal, topical (including buccal and sub-lingual), vaginal orparenteral (including intramuscular, subcutaneous and intravenous)administration, or for administration by inhalation or insufflation.Preferably, administration is intravenous. The formulations areoptionally packaged in discrete dosage units.

Pharmaceutical formulations suitable for oral administration include,but are not limited to, capsules, cachets or tablets, each containing apredetermined amount of active ingredient. Suitable formulations alsoinclude, but are not limited to, powders, granules, solutions,suspensions and emulsions. The active ingredient is optionallyadministered as a bolus electuary or paste. Tablets and capsules fororal administration can contain conventional excipients, including, butnot limited to, binding agents, fillers, lubricants, disintegrant and/orwetting agents. A tablet can be made by compression or molding,optionally with one or more formulational ingredients. Compressedtablets can be prepared by compressing in a suitable machine the activeingredients in a free-flowing form, for example, a powder or granules,optionally mixed with a binder, lubricant, inert diluent, lubricating,surface active and/or dispersing agent. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets can be coatedaccording to methods well known in the art. Oral fluid preparations canbe in the form of, for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or can be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations can contain conventional additives, for example,suspending agents, emulsifying agents, non-aqueous vehicles (which caninclude edible oils), and/or preservatives. The tablets can optionallybe formulated so as to provide slow or controlled release of the activeingredient therein. A package of tablets can contain one tablet to betaken on each of the month.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions, optionally containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; as wellas aqueous and non-aqueous sterile suspensions including suspendingagents and/or thickening agents. The formulations can be presented inunit dose or multi-dose containers, for example as sealed ampoules andvials, and can be stored in a freeze-dried (lyophilized) condition,requiring only the addition of the sterile liquid carrier, for example,saline, water-for-injection, immediately prior to use. Alternatively,the formulations can be presented for continuous infusion.Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.

Formulations suitable for rectal administration include suppositorieswith standard carriers for example, cocoa butter or polyethylene glycol.Formulations suitable for topical administration in the mouth, forexample, buccally or sublingually, include lozenges, containing theactive ingredient in a flavored base, for example, sucrose and acacia ortragacanth, and pastilles, containing the active ingredient in a base,for example, gelatin and glycerin or sucrose and acacia. For intra-nasaladministration, the compounds of the invention can be used as a liquidspray, a dispersible powder, or in the form of drops. Drops can beformulated with an aqueous or non-aqueous base also including one ormore dispersing agents, solubilizing agents and/or suspending agents.

For administration by inhalation the compounds can be convenientlydelivered from an insufflator, nebulizer, pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs caninclude a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds can take the form of a dry powder composition, for example apowder mix of the compound and a suitable powder base, for example,lactose or starch. The powder composition can be presented in unitdosage form, for example, as capsules, cartridges, gelatin or blisterpacks, from which the powder can be administered with the aid of aninhalator or insufflators.

Other formulations include implantable devices and adhesive patcheswhich release a therapeutic agent.

When desired, the above described formulations, adapted to givesustained release of the active ingredient, can be employed. Thepharmaceutical compositions can also contain other active ingredients,including antimicrobial agents, immunosuppressants and/or preservatives.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention can include otheragents conventional in the art with regard to the type of formulation inquestion. For example, formulations suitable for oral administration caninclude flavoring agents.

Preferred unit dosage formulations contain an effective dose, as recitedbelow, or an appropriate fraction thereof, of the active ingredient.

For each of the aforementioned conditions, the compositions, e.g.,polypeptides and organic compounds, can be administered orally or viainjection at a dose ranging from about 0.1 to about 250 mg/kg per day.The dose range for adult humans is generally from about 5 mg to about17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferablyabout 100 mg to about 3 g/day. Tablets or other unit dosage forms ofpresentation provided in discrete units can conveniently contain anamount which is effective at such dosage or as a multiple of the same,for instance, units containing about 5 mg to about 500 mg, usually fromabout 100 mg to about 500 mg.

The dose employed will depend upon a number of factors, including theage and sex of the subject, the precise disorder being treated, and itsseverity. Also the route of administration can vary depending upon thecondition and its severity. In any event, appropriate and optimumdosages can be routinely calculated by those skilled in the art, takinginto consideration the above-mentioned factors.

Cancer Diagnosis:

By measuring the level of LY6K in a subject-derived biological sample,the occurrence of cancer or a predisposition to develop cancer in asubject can be determined. Preferably, cancer is either of esophagealand lung cancer, or both. Accordingly, the present invention involvesdetermining (e.g., measuring) the level of LY6K in a biological sample.

By measuring the level of LY6K in subject-derived blood samples, theoccurrence of lung cancer or esophageal cancer, or a predisposition todevelop lung cancer or esophageal cancer in a subject can be determined.Alternatively, according to the present invention, an intermediateresult for examining the condition of a subject may be provided. Suchintermediate result may be combined with additional information toassist a doctor, nurse, or other practitioner to determine that asubject suffers from lung cancer or esophageal cancer. Further, thepresent invention relates to a method for screening a person who isrequired to be further diagnosed for lung cancer or esophageal cancer.After the screening, persons indicating positive result are recommendedto be submitted further screening test, or medical treatment to confirmwhether they truly suffer from lung cancer or esophageal cancer.Accordingly, the present invention also provides LY6K protein as bloodtumor marker for diagnosing or screening of either of esophageal andlung cancer, or both.

Alternatively, the present invention may be used to detect cancerouscells in a subject-derived tissue, and provide a doctor with usefulinformation to determine that the subject suffers from lung cancer oresophageal cancer. Accordingly, the present invention involvesdetermining (e.g., measuring) the level of LY6K in subject derivedsamples, such as blood samples. In the present invention, a method fordiagnosing lung cancer or esophageal cancer also includes a method fortesting or detecting lung cancer or esophageal cancer. Alternatively, inthe present invention, diagnosing lung cancer or esophageal cancer alsorefers to showing a suspicion, risk, or possibility of lung cancer oresophageal cancer in a subject.

Any blood samples may be used for determining the level of LY6K so longas either the LY6K gene or the LY6K protein can be detected in thesamples. Preferably, the blood samples includes whole blood, serum, andplasma.

In the present invention, the “level of LY6K in blood samples” refers tothe concentration of LY6K present in the blood after correcting thecorpuscular volume in the whole blood. One of skill will recognize thatthe percentage of corpuscular volume in the blood varies greatly betweenindividuals. For example, the percentage of erythrocytes in the wholeblood is very different between men and women. Furthermore, differencesbetween individuals cannot be ignored. Therefore, the apparentconcentration of a substance in the whole blood which includescorpuscular components varies greatly depending on the percentage ofcorpuscular volume. For example, even if the concentration in the serumis the same, the measured value for a sample with a large amount ofcorpuscular component will be lower than the value for a sample with asmall amount of corpuscular component. Therefore, to compare themeasured values of components in the blood, values for which thecorpuscular volume has been corrected are usually used.

For example, by measuring components in the blood using, as samples,serum or plasma obtained by separating blood cells from the whole blood,measured values from which the effect from the corpuscular volume hasbeen removed can be obtained. Therefore, the level of LY6K in thepresent invention can usually be determined as a concentration in theserum or plasma. Alternatively, it may first be measured as aconcentration in the whole blood, then the effect from the corpuscularvolume may be corrected. Methods for measuring a corpuscular volume in awhole blood sample are known.

Subjects diagnosed with lung cancer or esophageal cancer according tothe present methods are preferably mammals and include humans, non-humanprimates, mice, rats, dogs, cats, horses and cows. A preferable subjectof the present invention is a human. In the present invention, a subjectmay be a healthy individual or a patient suspected of having either oflung cancer and esophagus cancer, or both. The patient may be diagnosedby the present invention to facilitate clinical decision-making. Inanother embodiment, the present invention may also be applied to healthyindividuals for screening of either of lung cancer and esophagus cancer,or both.

In one embodiment of the present invention, the level of LY6K isdetermined by measuring the quantity or concentration of LY6K protein inblood samples. Methods for determining the quantity of the LY6K proteinin blood samples include immunoassay methods.

In the diagnostic methods of the present invention, the bloodconcentration of CEA or CYFRA 21-1 or both may be determined, inaddition to the blood concentration of LY6K, to detect lung cancerand/or esophageal cancer. Therefore, the present invention providesmethods for diagnosing either or both of lung cancer and esophagealcancer, in which the cancers are detected when either the bloodconcentration of LY6K or the blood concentration of CYFRA 21-1, or bothof them, are higher as compared with healthy individuals. Similarly, theprevent invention provides methods for diagnosing either or both of lungcancer and esophageal cancer, in which the cancers are detected wheneither the blood concentration of LY6K or the blood concentration ofCEA, or both of them, are higher as compared with healthy individuals.Alternatively, either or both of lung cancer and esophageal cancer isdetected when at least one of blood concentration of LY6K, CYFRA 21-1,and CEA is higher as compared with healthy individuals.

CEA is associated with tumors and the developing fetus. Although CEA wasfirst identified in colon cancer, elevated CEA levels have been found ina variety of cancers apart from colonic, including pancreatic, gastric,lung, and breast cancers. The best use of CEA is as a tumor marker forcancers of the gastrointestinal tract. CYFRA 21-1 measures solublecytokeratin-19 fragments in serum, and is a useful marker for lungcarcinoma, especially squamous cell carcinoma. CYFRA 21-1 is a uniquetumor marker that uses two different monoclonal antibodies whichrecognize the divergent epitope on the N- or C-terminal region of domain2 of cytokeratin 19 fragment, respectively.

In the present invention, a novel serological marker for lung cancer oresophageal cancer, LY6K, is provided. Improvement in the sensitivity ofdiagnostic or detection methods for lung cancer or esophageal cancer maybe achieved by the present invention. Namely, the present inventionprovides a method for diagnosing lung cancer or esophageal cancer in asubject, including the steps of:

(a) collecting a blood sample from a subject to be diagnosed;

(b) determining a level of LY6K in the blood sample;

(c) comparing the LY6K level determined in step (b) with that of anormal control wherein a high LY6K level in the blood sample, comparedto the normal control, indicates that the subject suffers from lungcancer or esophageal cancer.

In preferable embodiments, the diagnostic or detection method of thepresent invention may further include the steps of:

(e) determining a level of either or both of CEA and CYFRA21-1 in theblood sample;

(f) comparing the either or both of CEA and CYFRA21-1 level determinedin step (e) with that of a normal control; and

(g) judging that high levels of LY6K and either or both of CEA andCYFRA21-1 in the blood sample, compared with the normal control,indicate that the subject suffers from lung cancer and/or esophagealcancer.

Furthermore, method of the measuring targets includes the combination ofLY6K and other cancer-associated proteins in biological sample fromsubject, e.g., CEA and CYFRA21-1. A high level of LY6K expression wasassociated with poor prognosis of patients with NSCLC (P=0.0026) as wellas ESCC (P=0.0455), and multivariate analysis confirmed its independentprognostic value for NSCLC (P=0.0201). The proportion of the serumLY6K-positive cases was 33.9% of NSCLC and 32.1% of ESCC, while only4.1% of healthy volunteers were falsely diagnosed as positive. Theproportion of the serum CEA-positive case was 39.8% of NSCLC, and theproportion of serum CYFRA 21-1-positive case was 39.8 of NSCLC. On theother hand, a combined assay using both LY6K and carcinoembryonicantigen (CEA) judged 64.7% of the lung adenocarcinoma patients aspositive while 9.5% of healthy volunteers were falsely diagnosed. Theuse of both LY6K and CYFRA 21-1 increased sensitivity to detect lungsquamous-cell carcinomas up to 70.4%, while false positive rate wereonly 6.8%. The sensitivity for detection of lung cancer or esophagealcancer may be significantly improved by combining LY6K with CEA and/orCYFRA 21-1. In the preferable embodiments, a patient with positiveresults of LY6K with CEA and/or CYFRA 21-1 with may be judged to have ahigh risk of lung cancer or esophageal cancer. The use of a combinationof LY6K with CEA and/or CYFRA21-1 as a serological marker for lungcancer and esophageal cancer is novel.

Accordingly, the present invention provides for great improvements inthe sensitivity of assays for detecting lung cancer or esophageal cancerin patients, as compared to determinations based on results of measuringCEA or CYFRA 21-1 alone. While not wishing to be bound by theory, it isbelieved that the fact that the group of CEA-positive or CYFRA21-1-positive patients and the group of LY6K-positive patients do notmatch completely is behind this marked improvement. This fact is furtherdescribed specifically below.

First, among patients who, as a result of CEA or CYFRA 21-1measurements, were determined to have a lower value than a standardvalue (i.e. not to have lung cancer or esophageal cancer), there isactually a certain percentage of patients that have lung cancer oresophageal cancer. Such patients are referred to as CEA- or CYFRA21-1-false negative patients. By combining a determination based on CEAor CYFRA 21-1 with a determination based on LY6K, patients whose LY6Kvalue is above the standard value can be found from among the CEA- orCYFRA 21-1-false negative patients. That is, from among patients falselydetermined to be “negative” due to a low blood concentration of CEA orCYFRA 21-1, the present invention provides a means to identify patientsactually having lung cancer or esophageal cancer. The sensitivity fordetecting lung cancer or esophageal cancer patients is thus improved bythe present invention. Generally, simply combining the results fromdeterminations using multiple markers may increase the detectionsensitivity, but on the other hand, it often causes a decrease inspecificity. However, by determining the best balance betweensensitivity and specificity, the present invention has determined acharacteristic combination that can increase the detection sensitivitywithout compromising the specificity.

In the context of the present invention, in order to consider theresults of CEA and

CYFRA 21-1 measurements at the same time, for example, the bloodconcentration of CEA or CYFRA 21-1 may be measured and compared withstandard values, in the same way as for the aforementioned comparisonbetween the measured values and standard values of LY6K. For example,how to measure the blood concentration of CEA or CYFRA 21-1 and compareit to standard values are already known. Moreover, ELISA kits for CEAand CYFRA 21-1 are also commercially available. These methods describedin known reports can be used in the method of the present invention fordiagnosing or detecting lung cancer or esophageal cancer.

In the context of the present invention, the standard value of the bloodconcentration of LY6K can be determined statistically. For example, theblood concentration of LY6K in healthy individuals can be measured todetermine the standard blood concentration of LY6K statistically. When astatistically sufficient population is gathered, a value in the range oftwice or three times the standard deviation (S.D.) from the mean valueis often used as the standard value. Therefore, values corresponding tothe mean value+2×S.D. or mean value+3×S.D. may be used as standardvalues. The standard values set as described theoretically include 90%and 99.7% of healthy individuals, respectively.

Alternatively, standard values can also be set based on the actual bloodconcentration of the LY6K protein in lung cancer or esophageal cancerpatients. Generally, standard values set this way minimize thepercentage of false positives, and are selected from a range of valuessatisfying conditions that can maximize detection sensitivity. Herein,the percentage of false positives refers to a percentage, among healthyindividuals, of patients whose blood concentration of LY6K is judged tobe higher than a standard value. On the contrary, the percentage, amonghealthy individuals, of patients whose blood concentration of LY6K isjudged to be lower than a standard value indicates specificity. That is,the sum of the false positive percentage and the specificity isalways 1. The detection sensitivity refers to the percentage of patientswhose blood concentration of LY6K is judged to be higher than a standardvalue, among all lung cancer or esophageal cancer patients within apopulation of individuals for whom the presence of lung cancer oresophageal cancer has been determined.

Furthermore, in the context of the present invention, the percentage oflung cancer or esophageal cancer patients among patients whose LY6Kconcentration was judged to be higher than a standard value representsthe positive predictive value. On the other hand, the percentage ofhealthy individuals among patients whose LY6K concentration was judgedto be lower than a standard value represents the negative predictivevalue. The relationship between these values is summarized in Table 1 asbelow. As the relationship shown below indicates, each of the values forsensitivity, specificity, positive predictive value, and negativepredictive value, which are indexes for evaluating the diagnosticaccuracy for lung cancer or esophageal cancer, varies depending on thestandard value for judging the level of the blood concentration of LY6K.

TABLE 1 Blood Lung cancer or concentration esophageal cancer Healthy ofLY6K patients individuals High a: True positive b: False positivePositive predictive value a/(a + b) Low c: False negative d: Truenegative Negative predictive value d/(c + d) Sensitivity Specificitya/(a + c) d/(b + d)

As mentioned previously, a standard value is usually set such that thefalse positive ratio is low and the sensitivity is high. However, asalso apparent from the relationship shown above, there is a trade-offbetween the false positive ratio and sensitivity. That is, if thestandard value is decreased, the detection sensitivity increases.However, since the false positive ratio also increases, it is difficultto satisfy the conditions to have a “low false positive ratio”.Considering this situation, for example, values that give the followingpredicted results may be selected as the preferable standard values inthe present invention.

Standard values for which the false positive ratio is 50% or less (thatis, standard values for which the specificity is not less than 50%).

Standard values for which the sensitivity is not less than 20%.

In the present invention, the standard values can be set using areceiver operating characteristic (ROC) curve. A ROC curve is a graphthat shows the detection sensitivity on the vertical axis and the falsepositive ratio (that is, “1—specificity”) on the horizontal axis. In thepresent invention, an ROC curve can be obtained by plotting the changesin the sensitivity and the false positive ratio, which were obtainedafter continuously varying the standard value for determining thehigh/low degree of the blood concentration of LY6K.

The “standard value” for obtaining the ROC curve is a value temporarilyused for the statistical analyses. The “standard value” for obtainingthe ROC curve can generally be continuously varied within a range thatallows to cover all selectable standard values. For example, thestandard value can be varied between the smallest and largest measuredLY6K values in an analyzed population.

Based on the obtained ROC curve, a preferable standard value to be usedin the present invention can be selected from a range that satisfies theabove-mentioned conditions. Alternatively, a standard value can beselected based on an ROC curve produced by varying the standard valuesfrom a range that includes most of the measured LY6K values.

LY6K in the blood can be measured by any conventional method suitablefor quantitating proteins. For example, immunoassay, liquidchromatography, surface plasmon resonance (SPR), mass spectrometry, orthe like can be used in the context of the present invention. In massspectrometry, proteins can be quantitated by using a suitable internalstandard. For example, isotope-labeled LY6K can be used as the internalstandard. The concentration of LY6K in the blood can be determined fromthe peak intensity of LY6K in the blood and that of the internalstandard. Generally, the matrix-assisted laser desorption/ionization(MALDI) method is used for mass spectrometry of proteins. With ananalysis method that uses mass spectrometry or liquid chromatography,LY6K can also be analyzed simultaneously with other tumor markers (e.g.,CEA or CYFRA 21-1).

A preferable method for measuring LY6K in the context of the presentinvention is the immunoassay. The amino acid sequence of LY6K is known(Genbank Accession Number HSJ001348, NM_(—)017527). The amino acidsequence of LY6K is shown in SEQ ID NO: 2, and the nucleotide sequenceof the cDNA encoding it is shown in SEQ ID NO: 1. Therefore, thoseskilled in the art can prepare antibodies by synthesizing necessaryimmunogens based on the amino acid sequence of LY6K. The peptide used asimmunogen can be easily synthesized using a peptide synthesizer. Thesynthetic peptide can be used as an immunogen by linking it to a carrierprotein.

Keyhole limpet hemocyanin, myoglobin, albumin, and the like can be usedas the carrier protein. Preferable carrier proteins are KLH, bovineserum albumin, and such. The maleimidobenzoyl-N-hydrosuccinimide estermethod (hereinafter abbreviated as the MB S method) and the like aregenerally used to link synthetic peptides to carrier proteins.

Specifically, a cysteine is introduced into the synthetic peptide andthe peptide is crosslinked to KLH by MBS using the cysteine's SH group.The cysteine residue may be introduced at the N-terminus or C-terminusof the synthesized peptide.

Alternatively, LY6K can be prepared using the nucleotide sequence ofLY6K (Genbank Accession Number HSJ001348, NM_(—)017527), or a portionthereof. DNAs having the necessary nucleotide sequence can be clonedusing mRNAs prepared from LY6K-expressing tissues. Alternatively,commercially available cDNA libraries can be used as the cloning source.The obtained genetic recombinants of LY6K, or fragments thereof, canalso be used as the immunogen. LY6K recombinants expressed in thismanner are preferable as the immunogen for obtaining the antibodies usedin the present invention.

Immunogens obtained in this manner are mixed with a suitable adjuvantand used to immunize animals. Known adjuvants include Freund's completeadjuvant (FCA) and incomplete adjuvant. The immunization procedure isrepeated at appropriate intervals until an increase in the antibodytiter is confirmed. There are no particular limitations on the immunizedanimals in the present invention. Specifically, animals commonly usedfor immunization such as mice, rats, or rabbits can be used.

When obtaining the antibodies as monoclonal antibodies, animals that areadvantageous for their production may be used. For example, in mice,many myeloma cell lines for cell fusion are known, and techniques forestablishing hybridomas with a high probability are already well known.Therefore, mice are a desirable immunized animal to obtain monoclonalantibodies.

Furthermore, the immunization treatments are not limited to in vitrotreatments. Methods for immunologically sensitizing culturedimmunocompetent cells in vitro can also be employed. Antibody-producingcells obtained by these methods are transformed and cloned. Methods fortransforming antibody-producing cells to obtain monoclonal antibodiesare not limited to cell fusion. For example, methods for obtainingclonable transformants by virus infection are known.

Hybridomas that produce the monoclonal antibodies used in the presentinvention can be screened based on their reactivity to LY6K.Specifically, antibody-producing cells are first selected by using as anindex the binding activity toward LY6K, or a domain peptide thereof,that was used as the immunogen. Positive clones that are selected bythis screening are subcloned as necessary.

The monoclonal antibodies to be used in the present invention can beobtained by culturing the established hybridomas under suitableconditions and collecting the produced antibodies. When the hybridomasare homohybridomas, they can be cultured in vivo by inoculating themintraperitoneally in syngeneic animals. In this case, monoclonalantibodies are collected as ascites fluid. When heterohybridomas areused, they can be cultured in vivo using nude mice as a host.

In addition to in vivo cultures, hybridomas are also commonly culturedex vivo, in a suitable culture environment. For example, basal mediasuch as RPMI 1640 and DMEM are generally used as the medium forhybridomas. Additives such as animal sera can be added to these media tomaintain the antibody-producing ability to a high level. When hybridomasare cultured ex vivo, the monoclonal antibodies can be collected as aculture supernatant. Culture supernatants can be collected by separatingfrom cells after culturing, or by continuously collecting whileculturing using a culture apparatus that uses a hollow fiber.

Monoclonal antibodies used in the context of the present invention canbe prepared from monoclonal antibodies collected as ascites fluid orculture supernatants, by separating immunoglobulin fractions bysaturated ammonium sulfate precipitation and further purifying by gelfiltration, ion exchange chromatography, or such. In addition, if themonoclonal antibodies are IgGs, purification methods based on affinitychromatography with a protein A or protein G column are effective.

On the other hand, to obtain antibodies useful in the context of thepresent invention as polyclonal antibodies which recognize amino acidsequence comprising SEQ ID NO: 18 or 19, blood can be drawn from animalswhose antibody titer increased after immunization, and the serum isseparated to obtain an anti-serum. Immunoglobulins are purified fromanti-sera by known methods to prepare the antibodies used in the presentinvention. LY6K-specific antibodies can be prepared by combiningimmunoaffinity chromatography which uses LY6K as a ligand withimmunoglobulin purification.

When antibodies against LY6K contact LY6K, the antibodies bind to theantigenic determinant (epitope) that the antibodies recognize through anantigen-antibody reaction. Especially, the epitope comprises SEQ ID NO:18 or 19. The binding of antibodies to antigens can be detected byvarious immunoassay principles. Immunoassays can be broadly categorizedinto heterogeneous analysis methods and homogeneous analysis methods. Tomaintain the sensitivity and specificity of immunoassays to a highlevel, monoclonal antibodies are preferred. Methods of the presentinvention for measuring LY6K by various immunoassay formats aredescribed in further detail below.

First, methods for measuring LY6K using a heterogeneous immunoassay aredescribed. In heterogeneous immunoassays, a mechanism for detectingantibodies that bound to LY6K after separating them from those that didnot bind to LY6K is required.

To facilitate the separation, immobilized reagents are generally used.For example, a solid phase onto which antibodies recognizing LY6K havebeen immobilized is first prepared (immobilized antibodies). LY6K ismade to bind to these, and secondary antibodies are further reactedthereto.

When the solid phase is separated from the liquid phase and furtherwashed, as necessary, secondary antibodies remain on the solid phase inproportion to the concentration of LY6K. By labeling the secondaryantibodies, LY6K can be quantitated by measuring the signal derived fromthe label.

Any method may be used to bind the antibodies to the solid phase. Forexample, antibodies can be physically adsorbed to hydrophobic materialssuch as polystyrene. Alternatively, antibodies can be chemically boundto a variety of materials having functional groups on their surfaces.Furthermore, antibodies labeled with a binding ligand can be bound to asolid phase by trapping them using a binding partner of the ligand.Combinations of a binding ligand and its binding partner includeavidin-biotin and such. The solid phase and antibodies can be conjugatedat the same time or before the reaction between the primary antibodiesand LY6K.

Similarly, the secondary antibodies do not need to be directly labeled.That is, they can be indirectly labeled using antibodies againstantibodies or using binding reactions such as that of avidin-biotin.

The concentration of LY6K in a sample is determined based on the signalintensities obtained using standard samples with known LY6Kconcentrations.

Any antibody can be used as the immobilized antibody and secondaryantibody for the heterogeneous immunoassays mentioned above, so long asit is an antibody, or a fragment containing an antigen-binding sitethereof, that recognizes LY6K. Therefore, it may be a monoclonalantibody, a polyclonal antibody, or a mixture or combination of both.For example, a combination of monoclonal antibodies and polyclonalantibodies is a preferable combination in the present invention.Alternatively, when both antibodies are monoclonal antibodies, combiningmonoclonal antibodies recognizing different epitopes is preferable.

In the present invention, for example, a combination of antibodiesrecognizing LY6K at codons 23-109 (SEQ ID NO: 18) and 71-204 (SEQ ID NO:19) are preferable to detect LY6K with high specificity.

Since the antigens to be measured are sandwiched by antibodies, suchheterogenous immunoassays are called sandwich methods. Since sandwichmethods excel in the measurement sensitivity and the reproducibility,they are a preferable measurement principle in the present invention.

The principle of competitive inhibition reactions can also be applied tothe heterogeneous immunoassays. Specifically, they are immunoassaysbased on the phenomenon where LY6K in a sample competitively inhibitsthe binding between LY6K with a known concentration and an antibody. Theconcentration of LY6K in the sample can be determined by labeling LY6Kwith a known concentration and measuring the amount of LY6K that reacted(or did not react) with the antibody.

A competitive reaction system is established when antigens with a knownconcentration and antigens in a sample are simultaneously reacted to anantibody. Furthermore, analyses by an inhibitory reaction system arepossible when antibodies are reacted with antigens in a sample, andantigens with a known concentration are reacted thereafter. In bothtypes of reaction systems, reaction systems that excel in theoperability can be constructed by setting either one of the antigenswith a known concentration used as a reagent component or the antibodyas the labeled component, and the other one as the immobilized reagent.

Radioisotopes, fluorescent substances, luminescent substances,substances having an enzymatic activity, macroscopically observablesubstances, magnetically observable substances, and such are used inthese heterogeneous immunoassays. Specific examples of these labelingsubstances are shown below.

Substances having an enzymatic activity:

peroxidase,

alkaline phosphatase,

urease, catalase,

glucose oxidase,

lactate dehydrogenase, or

amylase, etc.

Fluorescent substances:

fluorescein isothiocyanate,

tetramethylrhodamine isothiocyanate,

substituted rhodamine isothiocyanate, or

dichlorotriazine isothiocyanate, etc.

Radioisotopes:

tritium,

¹²⁵I, or

¹³¹I, etc.

Among these, non-radioactive labels such as enzymes are an advantageouslabel in terms of safety, operability, sensitivity, and such. Enzymaticlabels can be linked to antibodies or to LY6K by known methods such asthe periodic acid method or maleimide method.

As the solid phase, beads, inner walls of a container, fine particles,porous carriers, magnetic particles, or such are used. Solid phasesformed using materials such as polystyrene, polycarbonate,polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride,nylon, polymethacrylate, latex, gelatin, agarose, glass, metal, ceramic,or such can be used. Solid materials in which functional groups tochemically bind antibodies and such have been introduced onto thesurface of the above solid materials are also known. Known bindingmethods, including chemical binding such as poly-L-lysine orglutaraldehyde treatment and physical adsorption, can be applied forsolid phases and antibodies (or antigens).

Although the steps of separating the solid phase from the liquid phaseand the washing steps are required in all heterogeneous immunoassaysexemplified herein, these steps can easily be performed using theimmunochromatography method, which is a variation of the sandwichmethod.

Specifically, antibodies to be immobilized are immobilized onto porouscarriers capable of transporting a sample solution by the capillaryphenomenon, then a mixture of a sample containing LY6K and labeledantibodies is deployed therein by this capillary phenomenon. Duringdeployment, LY6K reacts with the labeled antibodies, and when it furthercontacts the immobilized antibodies, it is trapped at that location. Thelabeled antibodies that do not react with LY6K pass through, withoutbeing trapped by the immobilized antibodies.

As a result, the presence of LY6K can be detected using, as an index,the signals of the labeled antibodies that remain at the location of theimmobilized antibodies. If the labeled antibodies are maintainedupstream in the porous carrier in advance, all reactions can beinitiated and completed by just dripping in the sample solutions, and anextremely simple reaction system can be constructed. In theimmunochromatography method, labeled components that can bedistinguished macroscopically, such as colored particles, can becombined to construct an analytical device that does not even require aspecial reader.

Furthermore, in the immunochromatography method, the detectionsensitivity for

LY6K can be adjusted. For example, by adjusting the detectionsensitivity near the cutoff value described below, the aforementionedlabeled components can be detected when the cutoff value is exceeded. Byusing such a device, whether a subject is positive or negative can bejudged very simply. By adopting a constitution that allows a macroscopicdistinction of the labels, necessary examination results can be obtainedby simply applying blood samples to the device for immunochromatography.

Various methods for adjusting the detection sensitivity of theimmunochromatography method are known. For example, a second immobilizedantibody for adjusting the detection sensitivity can be placed betweenthe position where samples are applied and the immobilized antibodies(Japanese Patent Application Kokai Publication No. (JP-A) H06-341989(unexamined, published Japanese patent application)). LY6K in the sampleis trapped by the second immobilized antibody while deploying from theposition where the sample was applied to the position of the firstimmobilized antibody for label detection. After the second immobilizedantibody is saturated, LY6K can reach the position of the firstimmobilized antibody located downstream. As a result, when theconcentration of LY6K included in the sample exceeds a predeterminedconcentration, LY6K bound to the labeled antibody is detected at theposition of the first immobilized antibody.

Next, homogeneous immunoassays are explained. As opposed toheterogeneous immunological assay methods that require a separation ofthe reaction solutions as described above, LY6K can also be measuredusing homogeneous analysis methods. Homogeneous analysis methods allowthe detection of antigen-antibody reaction products without theirseparation from the reaction solutions.

A representative homogeneous analysis method is the immunoprecipitationreaction, in which antigenic substances are quantitatively analyzed byexamining precipitates produced following an antigen-antibody reaction.Polyclonal antibodies are generally used for the immunoprecipitationreactions. When monoclonal antibodies are applied, multiple types ofmonoclonal antibodies that bind to different epitopes of LY6K arepreferably used. The products of precipitation reactions that follow theimmunological reactions can be macroscopically observed or can beoptically measured for conversion into numerical data.

The immunological particle agglutination reaction, which uses as anindex the agglutination by antigens of antibody-sensitized fineparticles, is a common homogeneous analysis method. As in theaforementioned immunoprecipitation reaction, polyclonal antibodies or acombination of multiple types of monoclonal antibodies can be used inthis method as well. Fine particles can be sensitized with antibodiesthrough sensitization with a mixture of antibodies, or they can beprepared by mixing particles sensitized separately with each antibody.Fine particles obtained in this manner gives matrix-like reactionproducts upon contact with LY6K. The reaction products can be detectedas particle aggregation. Particle aggregation may be macroscopicallyobserved or can be optically measured for conversion into numericaldata.

Immunological analysis methods based on energy transfer and enzymechanneling are known as homogeneous immunoassays. In methods utilizingenergy transfer, different optical labels having a donor/acceptorrelationship are linked to multiple antibodies that recognize adjacentepitopes on an antigen. When an immunological reaction takes place, thetwo parts approach and an energy transfer phenomenon occurs, resultingin a signal such as quenching or a change in the fluorescencewavelength. On the other hand, enzyme channeling utilizes labels formultiple antibodies that bind to adjacent epitopes, in which the labelsare a combination of enzymes having a relationship such that thereaction product of one enzyme is the substrate of another. When the twoparts approach due to an immunological reaction, the enzyme reactionsare promoted; therefore, their binding can be detected as a change inthe enzyme reaction rate.

In the present invention, blood for measuring LY6K can be prepared fromblood drawn from patients. Preferable blood samples are the serum orplasma. Serum or plasma samples can be diluted before the measurements.Alternatively, the whole blood can be measured as a sample and theobtained measured value can be corrected to determine the serumconcentration. For example, concentration in whole blood can becorrected to the serum concentration by determining the percentage ofcorpuscular volume in the same blood sample.

In a preferred embodiment, the immunoassay is an ELISA. The presentinvention further provides sandwich ELISA to detect serum LY6K inpatients with lung cancer or esophageal cancer.

The LY6K level in the blood samples is then compared with an LY6K levelassociated with a reference sample such as a normal control sample. Thephrase “normal control level” refers to the level of LY6K typicallyfound in a blood sample of a population not suffering from lung canceror esophageal cancer. The reference sample is preferably of a similarnature to that of the test sample. For example, if the test sample iscomposed of patient serum, the reference sample should also be serum.The LY6K level in the blood samples from control and test subjects maybe determined at the same time or, alternatively, the normal controllevel may be determined by a statistical method based on the resultsobtained by analyzing the level of LY6K in samples previously collectedfrom a control group.

The LY6K level may also be used to monitor the course of treatment oflung cancer or esophageal cancer. In this method, a test blood sample isprovided from a subject undergoing treatment for lung cancer oresophageal cancer. Preferably, multiple test blood samples are obtainedfrom the subject at various time points before, during, or after thetreatment. The level of LY6K in the post-treatment sample may then becompared with the level of LY6K in the pre-treatment sample or,alternatively, with a reference sample (e.g., a normal control level).For example, if the post-treatment LY6K level is lower than thepre-treatment LY6K level, one can conclude that the treatment wasefficacious. Likewise, if the post-treatment LY6K level is similar tothe normal control LY6K level, one can also conclude that the treatmentwas efficacious.

An “efficacious” treatment is one that leads to a reduction in the levelof LY6K or a decrease in size, prevalence, or metastatic potential oflung cancer or esophageal cancer in a subject. When a treatment isapplied prophylactically, “efficacious” means that the treatment retardsor prevents occurrence of lung cancer or esophageal cancer or alleviatesa clinical symptom of lung cancer or esophageal cancer. The assessmentof lung cancer or esophageal cancer can be made using standard clinicalprotocols. Furthermore, the efficaciousness of a treatment can bedetermined in association with any known method for diagnosing ortreating lung cancer or esophageal cancer. For example, lung cancer oresophageal cancer is routinely diagnosed histopathologically or byidentifying symptomatic anomalies.

Therefore, the possibility that a patient judged to have lung cancer oresophageal cancer based on LY6K or a combination of LY6K with CEA and/orCYFRA 21-1 can be easily ruled out.

Components used to carry out the diagnosis of cancers such as lungcancer and esophageal cancer according to the present invention can becombined in advance and supplied as a testing kit. Accordingly, thepresent invention provides a kit for detecting lung cancer or esophagealcancer, including:

(i) an immunoassay reagent for determining a level of LY6K in a bloodsample; and

(ii) a positive control sample for LY6K.

In the preferable embodiments, the kit of the present invention mayfurther include:

(iii) an immunoassay reagent for determining either of the levels of CEAand CYFRA 21-1 or both in a blood sample; and

(iv) a positive control sample for CEA or CYFRA 21-1.

The reagents for the immunoassays which constitute a kit of the presentinvention may include reagents necessary for the various immunoassaysdescribed above. Specifically, the reagents for the immunoassays includean antibody that recognizes the substance to be measured. Especially,the antibody recognizes amino acid sequence comprising SEQ ID NO: 18 or19. The antibody can be modified depending on the assay format of theimmunoassay. ELISA can be used as a preferable assay format of thepresent invention. In ELISA, for example, a first antibody immobilizedonto a solid phase and a second antibody having a label are generallyused.

Therefore, the immunoassay reagents for ELISA can include a firstantibody immobilized onto a solid phase carrier. Fine particles or theinner walls of a reaction container can be used as the solid phasecarrier. Magnetic particles can be used as the fine particles.Alternatively, multi-well plates such as 96-well microplates are oftenused as the reaction containers. Containers for processing a largenumber of samples, which are equipped with wells having a smaller volumethan in 96-well microplates at a high density, are also known. In thepresent invention, the inner walls of these reaction containers can beused as the solid phase carriers.

The immunoassay reagents for ELISA may further include a second antibodyhaving a label. The second antibody for ELISA may be an antibody ontowhich an enzyme is directly or indirectly linked. Methods for chemicallylinking an enzyme to an antibody are known. For example,immmunoglobulins can be enzymatically cleaved to obtain fragments thatinclude the variable regions. By reducing the —SS— bonds contained inthese fragments to —SH groups, bifunctional linkers can be attached. Bylinking an enzyme to the bifunctional linkers in advance, enzymes can belinked to the antibody fragments.

Alternatively, to indirectly link an enzyme, for example, theavidin-biotin binding can be used. That is, an enzyme can be indirectlylinked to an antibody by contacting a biotinylated antibody with anenzyme to which avidin has been attached. In addition, an enzyme can beindirectly linked to a second antibody using a third antibody which isan enzyme-labeled antibody recognizing the second antibody. For example,enzymes such as those exemplified above can be used as the enzymes tolabel the antibodies.

Kits of the present invention may include a positive control for LY6K. Apositive control for LY6K includes LY6K whose concentration has beendetermined in advance. For example, a control sample whose LY6Kconcentration is higher than the cut-off value may be used as thepositive control. Alternatively, preferable concentrations are, forexample, a concentration set as the standard value in a testing methodof the present invention. Further, a positive control having a higherconcentration can also be combined. The positive control for LY6K in thepresent invention can additionally include CEA or CYFRA 21-1 whoseconcentration has been determined in advance. A positive controlincluding LY6K and CEA and/or CYFRA 21-1 is preferable as the positivecontrol of the present invention.

Therefore, the present invention provides a positive control fordetecting cancers such as lung cancer and esophageal cancer whichincludes LY6K and CEA and/or CYFRA 21-1 at concentrations above a normalvalue. Alternatively, the present invention relates to the use of ablood sample comprising LY6K and CEA and/or CYFRA 21-1 at concentrationsabove a normal value in the production of a positive control for thedetection of lung cancer or esophageal cancer. It has been known thatCEA or CYFRA 21-1 can serve as an index for lung cancer or esophagealcancer; however, that LY6K can serve as an index for lung cancer oresophageal cancer is a novel finding obtained by the present invention.Therefore, positive controls including LY6K and CEA and/or CYFRA 21-1are novel. The positive controls of the present invention can beprepared by adding LY6K and CEA and/or CYFRA 21-1 at concentrationsabove a standard value to blood samples. For example, sera includingLY6K and CEA and/or CYFRA 21-1 at concentrations above a standard valueare preferable as the positive controls of the present invention.

The positive controls in the present invention are preferably in aliquid form. In the present invention, blood samples are used assamples. Therefore, samples used as controls also need to be in a liquidform. Alternatively, by dissolving a dried positive control with apredefined amount of liquid at the time of use, a control that gives thetested concentration can be prepared. By packaging, together with adried positive control, an amount of liquid necessary to dissolve it,the user can obtain the necessary positive control by just mixing them.LY6K used as the positive control can be a naturally-derived protein orit may be a recombinant protein. Not only positive controls, but alsonegative controls can be combined in the kits of the present invention.The positive controls or negative controls are used to verify that theresults indicated by the immunoassays are correct.

Method for Assessing the Prognosis of Cancer

According to the present invention, it was newly discovered that LY6Kexpression is significantly associated with poorer prognosis in patients(see FIG. 3B and D). Thus, the present invention provides a method fordetermining or assessing the prognosis of a patient with cancer, inparticular, esophageal and/or lung cancer, by detecting the expressionlevel of the LY6K gene in a biological sample of the patient; comparingthe detected expression level to a control level; and correlating anincreased expression level to the control level with an indication ofpoor prognosis (poor survival). Alternatively, according to the presentinvention, an intermediate result for determining or assessing theprognosis of a subject may be provided. Such intermediate result may becombined with additional information to assist a doctor, nurse, or otherpractitioner to determine or assess the prognosis of a patient withcancer. Alternatively, the present invention may be used to detectcancerous cells in a subject-derived tissue, and provide a doctor withuseful information to determine or assess the prognosis of a patientwith cancer.

Herein, the term “prognosis” refers to a forecast as to the probableoutcome of the disease as well as the prospect of recovery from thedisease as indicated by the nature and symptoms of the case.Accordingly, a less favorable, negative, poor prognosis is defined by alower post-treatment survival term or survival rate. Conversely, apositive, favorable, or good prognosis is defined by an elevatedpost-treatment survival term or survival rate.

The terms “assessing the prognosis” refer to the ability of predicting,forecasting or correlating a given detection or measurement with afuture outcome of cancer of the patient (e.g., malignancy, likelihood ofcuring cancer, survival, and the like). For example, a determination ofthe expression level of LY6K over time enables a predicting of anoutcome for the patient (e.g., increase or decrease in malignancy,increase or decrease in grade of a cancer, likelihood of curing cancer,survival, and the like).

In the context of the present invention, the phrase “assessing (ordetermining) the prognosis” is intended to encompass predictions andlikelihood analysis of cancer, progression, particularly cancerrecurrence, metastatic spread and disease relapse. The present methodfor assessing prognosis is intended to be used clinically in makingdecisions concerning treatment modalities, including therapeuticintervention, diagnostic criteria such as disease staging, and diseasemonitoring and surveillance for metastasis or recurrence of neoplasticdisease.

The patient-derived biological sample used for the method may be anysample derived from the subject to be assessed so long as the LY6K genecan be detected in the sample. Preferably, the biological sample is anesophageal and lung cell (a cell obtained from the esophagus and lung).Furthermore, the biological sample includes bodily fluids such assputum, blood, serum, or plasma. Moreover, the sample may be cellspurified from a tissue. The biological samples may be obtained from apatient at various time points, including before, during, and/or after atreatment.

According to the present invention, it was shown that the higher theexpression level of the LY6K gene measured in the patient-derivedbiological sample, the poorer the prognosis for post-treatmentremission, recovery, and/or survival and the higher the likelihood ofpoor clinical outcome. Thus, according to the present method, the“control level” used for comparison may be, for example, the expressionlevel of the LY6K gene detected before any kind of treatment in anindividual or a population of individuals who showed good or positiveprognosis of cancer, after the treatment, which herein will be referredto as “good prognosis control level”. Alternatively, the “control level”may be the expression level of the LY6K gene detected before any kind oftreatment in an individual or a population of individuals who showedpoor or negative prognosis of cancer, after the treatment, which hereinwill be referred to as “poor prognosis control level”. The “controllevel” is a single expression pattern derived from a single referencepopulation or from a plurality of expression patterns. Thus, the controllevel may be determined based on the expression level of the LY6K genedetected before any kind of treatment in a patient of cancer, or apopulation of the patients whose disease state (good or poor prognosis)is known. Preferably, cancer is esophageal or lung cancer. It ispreferable to use the standard value of the expression levels of theLY6K gene in a patient group with a known disease state. The standardvalue may be obtained by any method known in the art. For example, arange of mean+/−2S.D. or mean+/−3S.D. may be used as standard value.

The control level may be determined at the same time with the testbiological sample by using a sample(s) previously collected and storedbefore any kind of treatment from cancer patient(s) (control or controlgroup) whose disease state (good prognosis or poor prognosis) are known.

Alternatively, the control level may be determined by a statisticalmethod based on the results obtained by analyzing the expression levelof the LY6K gene in samples previously collected and stored from acontrol group. Furthermore, the control level can be a database ofexpression patterns obtained from previously tested cells. Moreover,according to an aspect of the present invention, the expression level ofthe LY6K gene in a biological sample may be compared to multiple controllevels, which control levels are determined from multiple referencesamples. It is preferred to use a control level determined from areference sample derived from a tissue type similar to that of thepatient-derived biological sample.

According to the present invention, a similarity in the expression levelof the LY6K gene relative to the good prognosis control level indicatesa more favorable prognosis of the patient and an increase in theexpression level relative to the good prognosis control level indicatesless favorable, poorer prognosis for post-treatment remission, recovery,survival, and/or clinical outcome. On the other hand, a decrease in theexpression level of the LY6K gene relative to the poor prognosis controllevel indicates a more favorable prognosis of the patient and asimilarity in the expression level relative to the poor prognosiscontrol level indicates less favorable, poorer prognosis forpost-treatment remission, recovery, survival, and/or clinical outcome.

An expression level of the LY6K gene in a biological sample can beconsidered altered when the expression level differs from the controllevel by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.Alternatively, an expression level of the LY6K gene in a biologicalsample can be considered altered, when the expression level is increasedor decreased to the control level at least 10%, 20%, 30%, 40%, 50%, 60%,80%, 90%, or more.

The difference in the expression level between the test biologicalsample and the control level can be normalized to a control, e.g.,housekeeping gene. For example, polynucleotides whose expression levelsare known not to differ between the cancerous and non-cancerous cells,including those coding for beta-actin, glyceraldehyde 3-phosphatedehydrogenase, and ribosomal protein P1, may be used to normalize theexpression levels of the LY6K gene.

The expression level may be determined by detecting the gene transcriptin the patient-derived biological sample using techniques well known inthe art. The gene transcripts detected by the present method includeboth the transcription and translation products, such as mRNA andprotein.

For instance, the transcription product of the LY6K gene can be detectedby hybridization, e.g., Northern blot hybridization analyses, that usean LY6K gene probe to the gene transcript. The detection may be carriedout on a chip or an array. The use of an array is preferable fordetecting the expression level of a plurality of genes including theLY6K gene. As another example, amplification-based detection methods,such as reverse-transcription based polymerase chain reaction (RT-PCR)which use primers specific to the LY6K gene may be employed for thedetection (see Example). The LY6K gene-specific probe or primers may bedesigned and prepared using conventional techniques by referring to thewhole sequence of the LY6K gene (SEQ ID NO: 1). For example, the primersets (SEQ ID NOs: 3 and 4, 7 and 4) used in the Example may be employedfor the detection by RT-PCR, but the present invention is not restrictedthereto.

Specifically, a probe or primer used for the present method hybridizesunder stringent, moderately stringent, or low stringent conditions tothe mRNA of the LY6K gene. As used herein, the phrase “stringent(hybridization) conditions” refers to conditions under which a probe orprimer will hybridize to its target sequence, but to no other sequences.Stringent conditions are sequence-dependent and will be different underdifferent circumstances. Specific hybridization of longer sequences isobserved at higher temperatures than shorter sequences. Generally, thetemperature of a stringent condition is selected to be about 5 degreesC. lower than the thermal melting point (Tm) for a specific sequence ata defined ionic strength and pH. The Tm is the temperature (underdefined ionic strength, pH and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Since the target sequences are generallypresent at excess, at Tm, 50% of the probes are occupied at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30 degrees C. for short probes or primers(e.g., 10 to 50 nucleotides) and at least about 60 degrees C. for longerprobes or primers. Stringent conditions may also be achieved with theaddition of destabilizing agents, such as formamide.

Alternatively, the translation product may be detected for theassessment of the present invention. For example, the quantity of theLY6K protein may be determined. A method for determining the quantity ofthe protein as the translation product includes immunoassay methods thatuse an antibody specifically recognizing the LY6K protein. The antibodymay be monoclonal or polyclonal. Especially, the antibody recognizesamino acid sequence comprising SEQ ID NO: 18 or 19. Furthermore, anyfragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab′)2,Fv, etc.) of the antibody may be used for the detection, so long as thefragment retains the binding ability to the LY6K protein. Methods toprepare these kinds of antibodies for the detection of proteins are wellknown in the art, and any method may be employed in the presentinvention to prepare such antibodies and equivalents thereof.

As another method to detect the expression level of the LY6K gene basedon its translation product, the intensity of staining may be observedvia immunohistochemical analysis using an antibody against LY6K protein.Namely, the observation of strong staining indicates an increasedpresence of the LY6K protein and at the same time high expression levelof the LY6K gene.

Furthermore, the LY6K protein is known to have a cell proliferatingactivity.

Therefore, the expression level of the LY6K gene can be determined usingsuch cell proliferating activity as an index. For example, cells from abiological sample are prepared and cultured, and then by detecting thespeed of proliferation, or by measuring the cell cycle or the colonyforming ability, the expression level of the LY6K gene can bedetermined.

Moreover, in addition to the expression level of the LY6K gene, theexpression level of other esophageal and lung cell-associated genes, forexample, genes known to be differentially expressed in esophageal andlung cancer, may also be determined to improve the accuracy of theassessment. Examples of other lung cell-associated genes include, butare not limited to, those described in the WIPO Publication WO2004/031413, the entire contents of which are incorporated by referenceherein. In this publication, LY6K is referred to as URLC10.

The patient to be assessed for the prognosis of cancer according to themethod is preferably a mammal and includes human, non-human primate,mouse, rat, dog, cat, horse, and cow.

A Kit for Diagnosing Cancer and Assessing the Prognosis of Cancer

Kits:

The present invention provides a kit for diagnosing cancer or assessingthe prognosis of cancer, preferably, esophageal or lung cancer.Specifically, the kit contains at least one reagent for detecting theexpression of the LY6K gene in a patient-derived biological sample,which reagent may be selected from the group of:

(a) a reagent for detecting mRNA of the LY6K gene;

(b) a reagent for detecting the LY6K protein; and

(c) a reagent for detecting the biological activity of the LY6K protein.

Suitable reagents for detecting mRNA of the LY6K gene include nucleicacids that specifically bind to or identify the LY6K mRNA, such asoligonucleotides which have a complementary sequence to a part of theLY6K mRNA. These kinds of oligonucleotides are exemplified by primersand probes that are specific to the LY6K mRNA. These kinds ofoligonucleotides may be prepared based on methods well known in the art.If needed, the reagent for detecting the LY6K mRNA may be immobilized ona solid matrix. Moreover, more than one reagent for detecting the LY6KmRNA may be included in the kit.

On the other hand, suitable reagents for detecting the LY6K proteininclude antibodies to the LY6K protein. The antibody may be monoclonalor polyclonal, e.g., TM38 and MB44 which recognize LY6K at codons 23-109(SEQ ID NO: 18) and 71-204 (SEQ ID NO: 19), respectively. These twohighly LY6K-specific antibodies recognize different epitopes of the LY6Kprotein, and can be suitably used as the primary and secondaryantibodies in sandwich ELISA assays in the present invention.Furthermore, any fragmental or modified version (e.g., chimericantibody, scFv, Fab, F(ab′)2, Fv, etc.) of the antibody may be used asthe reagent, so long as the fragment retains the binding ability to theLY6K protein. Methods to prepare these kinds of antibodies for thedetection of proteins are well known in the art, and any method may beemployed in the present invention to prepare such antibodies andequivalents thereof. Furthermore, the antibody may be labeled withsignal generating molecules via direct linkage or an indirect labelingtechnique. Labels and methods for labeling antibodies and detecting thebinding of antibodies to their targets are well known in the art and anylabels and methods may be employed for the present invention. Moreover,more than one reagent for detecting the LY6K protein may be included inthe kit.

Furthermore, when a cell expressing LY6K, the biological activity can bedetermined by, for example, measuring the cell proliferating activitydue to the expressed LY6K protein. For example, the cell is cultured inthe presence of a patient-derived biological sample, and then bydetecting the speed of proliferation, or by measuring the cell cycle orthe colony forming ability the cell proliferating activity of thebiological sample can be determined. If needed, the reagent fordetecting the LY6K mRNA may be immobilized on a solid matrix. Moreover,more than one reagent for detecting the biological activity of the LY6Kprotein may be included in the kit.

The kit may contain more than one of the aforementioned reagents.Furthermore, the kit may contain a solid matrix and reagent for bindinga probe against the LY6K gene or antibody against the LY6K protein, amedium and container for culturing cells, positive and negative controlreagents, and a secondary antibody for detecting an antibody against theLY6K protein. For example, tissue samples obtained from patient withgood prognosis or poor prognosis may serve as useful control reagents. Akit of the present invention may further include other materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts (e.g.,written, tape, CD-ROM, etc.) with instructions for use. These reagentsand such may be included in a container with a label. Suitablecontainers include bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic.

The assay format of the kit can be a Northern hybridization or asandwich ELISA, both of which are known in the art. See, for example,Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^(rd)Edition, 2001, Cold Spring Harbor Laboratory Press; and UsingAntibodies, supra.

For example, an LY6K detection reagent can be immobilized on a solidmatrix, for example a porous strip, to form at least one LY6K detectionsite. The measurement or detection region of the porous strip caninclude a plurality of sites, each containing a nucleic acid. A teststrip can also contain sites for negative and/or positive controls.Alternatively, control sites can be located on a separate strip from thetest strip. Optionally, the different detection sites can containdifferent amounts of immobilized nucleic acids, i.e., a higher amount inthe first detection site and lesser amounts in subsequent sites. Uponthe addition of test sample, the number of sites displaying a detectablesignal provides a quantitative indication of the amount of LY6K presentin the sample. The detection sites can be configured in any suitablydetectable shape and are typically in the shape of a bar or dot spanningthe width of a test strip.

As an embodiment of the present invention, when the reagent is a probeagainst the LY6K mRNA, the reagent may be immobilized on a solid matrix,such as a porous strip, to form at least one detection site. Themeasurement or detection region of the porous strip may include aplurality of sites, each containing a nucleic acid (probe). A test stripmay also contain sites for negative and/or positive controls.Alternatively, control sites may be located on a strip separated fromthe test strip. Optionally, the different detection sites may containdifferent amounts of immobilized nucleic acids, i.e., a higher amount inthe first detection site and lesser amounts in subsequent sites. Uponthe addition of test sample, the number of sites displaying a detectablesignal provides a quantitative indication of the amount of LY6K mRNApresent in the sample. The detection sites may be configured in anysuitably detectable shape and are typically in the shape of a bar or dotspanning the width of a test strip.

The kit of the present invention may further contain a positive controlsample or LY6K standard sample. The positive control sample of thepresent invention may be prepared by collecting LY6K positive bloodsamples and then those LY6K level are assayed. Alternatively, purifiedLY6K protein or polynucleotide may be added to LY6K free serum to formthe positive sample or the LY6K standard. In the present invention,purified LY6K may be recombinant protein. The LY6K level of the positivecontrol sample is, for example more than cut off value.

Furthermore, the present invention provides a kit containing at leastone reagent for detecting the expression of the LY6K gene and one ormore reagent for detecting the expression of other cancer-associatedproteins in a patient-derived biological sample. Suitable reagents fordetecting the other cancer-associated proteins include antibodies to theother cancer-associated proteins, e.g., ELISA. For example, the levelsof CEA in serum were measured by ELISA with a commercially availableenzyme test kit (HOPE Laboratories, Belmont, Calif.), according to thesupplier's recommendations, the levels of CYFRA 21-1 in serum weremeasured by ELISA with a commercially available kit (DRG, Marburg,Germany).

Hereinafter, the present invention is described in more detail byreference to the

Examples. However, the following materials, methods and examples onlyillustrate aspects of the invention and in no way are intended to limitthe scope of the present invention. As such, methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention.

Examples Example 1

Materials and Methods

Cell Lines:

The 5 human NSCLC cell lines used in this study included threeadenocarcinoma cell lines (ADCs; A427, LC319 and NCI-H1373), twosquamous-cell carcinoma cell lines (SCCs; RERF-LC-AI and NCI-H226)(Hammarstrom S. Semin Cancer Biol. 1999 April;9(2):67-81.). All cellswere grown in monolayers in appropriate media supplemented with 10%fetal calf serum (FCS) and were maintained at 37 degrees C. in anatmosphere of humidified air with 5% CO₂. Human small airway epithelialcells (SAEC) were grown in optimized medium (SAGM) purchased fromCambrex Bio Science Inc. Primary NSCLC and ESCC samples had beenobtained earlier with informed consent (Taniwaki M, et al, Int J Oncol.2006 September;29(3):567-75; Yamabuki T, et al, Int J Oncol. 2006June;28(6):1375-84; Ishikawa N, et al. Cancer Sci. 2006August;97(8):737-45.).

A total of 413 formalin-fixed samples of primary NSCLCs (259 ADCs, 113SCCs, 28 LCCs, 13 ASCs; 129 female and 284 male patients; median age of64.5 with a range of 26-84 years), and adjacent normal lung tissues, hadbeen obtained earlier along with clinicopathological data from patientsundergoing curative surgery.

A total of 271 formalin-fixed primary ESCCs (26 female and 245 malepatients; median age of 61.4+/−8.1 SD with a range of 38-77 years) andadjacent normal esophageal tissue samples had also been obtained frompatients undergoing curative surgery. NSCLC specimen and five tissues(heart, liver, lung, kidney, and testis) from post-mortem materials (2individuals with SCC) were also obtained.

The pathological stage was determined according to the classification ofthe Union

Internationale Controle Cancer (Travis W D, et al., World HealthOrganization International Histological classification of tumours1999.). This study and the use of all clinical materials mentioned wereapproved by individual institutional Ethical Committees.

Serum Samples:

Serum samples were obtained with informed consent from 74 healthyindividuals as controls (14 females and 60 males; median age 48.0+/−7.47SD with a range of 33-60 years), and from 65 non-neoplastic lung diseasepatients with chronic obstructive pulmonary disease (COPD) enrolled as apart of the Japanese Project for Personalized Medicine (BioBank Japan)or admitted to Hiroshima University Hospital (8 females and 57 males;median age of 66.0+/−5.92 SD with a range of 54-73 years). All of thesepatients were current and/or former smokers (The mean[+/−1SD] ofpack-year index (PYI) was 55.6+/−50.1 SD; PYI was defined as the numberof cigarette packs [20 cigarette per pack] consumed a day multiplied byyears).

Serum samples were also obtained from 112 NSCLC patients (40 females and72 males; median age 66.0+/−12.0 SD with a range of 30-84) and 81esophageal-cancer patients (12 females and 69 males; median age65.0+/−5.1 SD with a range of 37-74). These 112 NSCLC cases included 85ADCs and 27 SCCs. Samples were selected for the study on the basis ofthe following criteria:

(1) patients were newly diagnosed and previously untreated and

(2) their tumors were pathologically diagnosed as lung or esophagealcancers (stages I-IV). Serum was obtained at the time of diagnosis andstored at −150 degrees C.

Semi-Quantitative RT-PCR:

Total RNA was extracted from cultured cells and clinical tissues usingTrizol reagent (Life Technologies, Inc. Gaithersburg, Md.) according tothe manufacturer's protocol. Extracted RNAs and normal human-tissuepoly(A) RNAs were treated with DNase I (Roche Diagnostics, Basel,Switzerland) and then reverse-transcribed using oligo (dT)12-18 primerand SuperScript II reverse transcriptase (Life Technologies, Inc.). Thenucleotide sequences of the primers for the semi-quantitative RT-PCRexperiments are follows:

LY6K gene-specific primers 5′-ATTCGCTACTGCAATTTAGAGG-3′ (SEQ ID NO: 3)and 5′-GTTTAATGCAACAGGTGACAACG-3′, (SEQ ID NO: 4))beta-actin (ACTB)-specific primers 5′-GAGGTGATAGCATTGCTTTCG-3′(SEQ ID NO: 5) and 5′-CAAGTCAGTGTACAGGTAAGC-3′. (SEQ ID NO: 6))All PCR reactions involved initial denaturation at 94 degrees C. for 2min followed by 22 (for ACTB) or 30 cycles (for LY6K) of 94 degrees C.30 s, 58 degrees C. for 30 s, and 72 degrees C. for 60 s on a GeneAmpPCR system 9700 (Applied Biosystems, Foster City, Calif.).

Northern-Blot Analysis:

Human multiple-tissue blots (BD Biosciences, Palo Alto, Calif.) werehybridized with ₃₂P-labeled PCR products. PCR product of LY6K wasprepared as a probe by RT-PCR using primers5′-AGGGTGACAATAGAGTGTGGTGT-3′ (SEQ ID NO: 7) and5′-GTTTAATGCAACAGGTGACAACG-3′ (SEQ ID NO: 4). Prehybridization,hybridization, and washing were performed according to the supplier'srecommendations. The blots were autoradiographed with intensifyingscreens at −80 degrees C. for one week.

RNA Interference Assay:

A vector-based RNA interference (RNAi) system, psiH1BX3.0, had beenpreviously established to direct the synthesis of siRNAs in mammaliancells (Suzuki C, et al. Cancer Res. 2003 Nov. 1;63(21):7038-41; Kato T,et al. Cancer Res. 2005 Jul. 1;65(13):5638-46.). 10 mcg ofsiRNA-expression vector, using 30 mcl of Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.), was transfected into lung-cancer cell lines, whichover-expressed LY6K. The transfected cells were cultured for five daysin the presence of appropriate concentrations of geneticin (G418), andthen cell numbers and viability were measured by Giemsa staining andtriplicate MTT assays. The target sequences of the syntheticoligonucleotides for RNAi were as follows: control 1 (EGFP: enhancedgreen fluorescent protein (GFP) gene, a mutant of Aequorea victoriaGFP), 5′-GAAGCAGCACGACTTCTTC-3′ (SEQ ID NO: 8); control 2 (Scramble(SCR): chloroplast Euglena gracilis gene coding for 5S and 16S rRNAs),5′-GCGCGCTTTGTAGGATTCG-3′ (SEQ ID NO: 9);

LY6K siRNA-1 (si-LY6K-1), 5′-AACCTGACTGCGAGACAACGA-3′ (SEQ ID NO: 10)(at the position of 473-493 nt of SEQ ID NO: 1);

LY6K siRNA-2 (si-LY6K-2), 5′-AAGGAGGTGCAAATGGACAGA-3′ (SEQ ID NO: 11)(at the position if 586-606 nt of SEQ ID NO: 1). Down-regulation of LY6Kprotein expression by effective siRNA (si-LY6K-2), but not by the twocontrols or si-LY6K-1, was confirmed with western-blotting in the celllines used for this assay.

SEQ ID clone NO sequence si-LY6K-1 target 10 AACCTGACTGCGAGACAACGA oligo12 TCCCAACCTGACTGCGAGACAACGATTC sense AAGAGATCGTTGTCTCGCAGTCAGGTT oligo13 AAAAAACCTGACTGCGAGACAACGATCT antisense CTTGAATCGTTGTCTCGCAGTCAGGTThairpin 14 AACCTGACTGCGAGACAACGATTCAAGA GATCGTTGTCTCGCAGTCAGGTTsi-LY6K-2 target 11 AAGGAGGTGCAAATGGACAGA oligo 15TCCCAAGGAGGTGCAAATGGACAGATTC sense AAGAGATCTGTCCATTTGCACCTCCTT oligo 16AAAAAAGGAGGTGCAAATGGACAGATCT antisense CTTGAATCTGTCCATTTGCACCTCCTThairpin 17 AAGGAGGTGCAAATGGACAGATTCAAGA GATCTGTCCATTTGCACCTCCTT

Preparation of Anti-LY6K Polyclonal Antibody:

Two types of rabbit antibodies termed TM38 and MB44 specific for LY6Kwere raised by immunizing rabbits with 6-histidine fused human LY6Kprotein (codons 23-109 (SEQ ID NO: 18) and 71-204 (SEQ ID NO: 19),respectively), and purified with standard protocols using affinitycolumns (Affi-gel 10; Bio-Rad Laboratories, Hercules, Calif.) conjugatedwith the 6-histidine fused protein. On western blots, it was confirmedthat the antibodies were specific for LY6K, using lysates from NSCLCtissues and cell lines as well as normal lung tissues.

Western-Blotting:

An ECL western-blotting analysis system (GE Healthcare Bio-sciences,Piscataway, N.J.) was used. SDS-PAGE was performed in 7.5%polyacrylamide gels. PAGE-separated proteins were electro-blotted ontonitrocellulose membranes (GE Healthcare Bio-sciences) and incubated witha rabbit polyclonal anti-human LY6K antibody. A goat anti-rabbit IgG-HRPantibody (GE Healthcare Bio-sciences) was served as the secondaryantibodies for these experiments.

Immunohistochemistry and Tissue Microarray:

Tumor-tissue microarrays were constructed using 413 formalin-fixedprimary NSCLCs and 271 ESCCs, as published previously (Chin S F, et al.Mol Pathol. 2003 October;56(5):275-9; Callagy G, et al. Diagn MolPathol. 2003 March;12(1):27-34; Callagy G, et al. J Pathol. 2005February;205(3):388-96.). Briefly, the tissue area for sampling wasselected based on visual alignment with the corresponding HE-stainedsection on a slide. Three, four, or five tissue cores (diameter 0.6 mm;height 3-4 mm) taken from a donor tumor block were placed into arecipient paraffin block using a tissue microarrayer (BeecherInstruments, Sun Prairie, Wis.). A core of normal tissue was punchedfrom each case, and 5-micrometer sections of the resulting microarrayblock were used for immunohistochemical analysis.

To investigate the status of the LY6K protein in clinical lung-cancersamples that had been embedded in paraffin blocks, the sections werestained in the following manner. Briefly, a rabbit polyclonal anti-humanLY6K antibody (TM38) was added after blocking of endogenous peroxidaseand proteins. The sections were incubated with HRP-labeled anti-rabbitIgG as the secondary antibody. Substrate-chromogen was added and thespecimens were counterstained with hematoxylin.

Three independent investigators assessed LY6K positivitysemi-quantitatively without prior knowledge of clinicopathological data.The intensity of LY6K staining was evaluated using following criteria:strong positive (2+), dark brown staining in more than 50% of tumorcells completely obscuring membrane and cytoplasm; weak positive (1+),any lesser degree of brown staining appreciable in tumor cell membraneand cytoplasm; absent (scored as 0), no appreciable staining in tumorcells. Cases were accepted only as strongly positive if reviewersindependently defined them as such.

Statistical Analysis:

Contingency tables were used to analyze the relationship of LY6Kexpression levels and clinicopathological variables of NSCLC or ESCCpatients. Tumor-specific survival curves were calculated from the dateof surgery to the time of death related to NSCLC or ESCC, or to the lastfollow-up observation. Kaplan-Meier curves were calculated for eachrelevant variable and for LY6K expression; differences in survival timesamong patient subgroups were analyzed using the log-rank test.

Univariate and multivariate analyses were performed with the Coxproportional-hazard regression model to determine associations betweenclinicopathological variables and cancer-related mortality. First,associations were analyzed between death and possible prognostic factorsincluding age, gender, histological type, pT-classification, andpN-classification, taking into consideration one factor at a time.Second, multivariate Cox analysis was applied on backward (stepwise)procedures that always forced LY6K expression into the model, along withany and all variables that satisfied an entry level of a p value smallerthan 0.05. As the model continued to add factors, independent factorsdid not exceed an exit level of P<0.05.

ELISA:

Serum levels of LY6K were measured by sandwich-type ELISA which had beenoriginally constructed. In brief, for detection of soluble LY6K inserum, 96-well flexible microtiter plates (439454; NALGE NUNCInternational, Rochester, N.Y.) were coated with 2 ng/ml of capturingpolyclonal antibody to LY6K (TM38) overnight. Wells were blocked with200 mcl PBS (pH 7.4) containing 1% BSA, 5% sucrose, and 0.05% NaN3 for 2hours and then incubated for 2 hours with 3-fold diluted serum samplesin PBS (pH 7.4) containing 1% BSA. After washing with PBS (pH 7.4)containing 0.05% Tween 20, the wells were incubated for 2 hours with 200ng/ml of biotin-conjugated polyclonal anti-LY6K antibody (MB44),followed by reaction with avidin-conjugated peroxidase (P347; DakoCytomation, Glostrup, Denmark) for 30 minutes using a Substrate Reagent(R&D Systems).

To prepare biotinylating rabbit polyclonal antibodies to LY6K (MB44),the Biotin Labeling Kit-NH2 (LK03) was used according to the supplier'sprotocol (DOJINDO LABORATORIES, Kumamoto, Japan). The color reaction wasstopped by adding 100 mcl of 2N sulfuric acid. Color intensity wasdetermined by a photometer at a wavelength of 450 nm, with a referencewave-length of 570 nm. A standard curve was drawn for each plate usingrecombinant LY6K proteins as a reference. Levels of CEA in serum weremeasured by ELISA with a commercially available enzyme test kit (HOPELaboratories, Belmont, Calif.), according to the supplier'srecommendations. Levels of CYFRA 21-1 in serum were measured by ELISAwith a commercially available kit (DRG, Marburg, Germany).

Differences in the levels of LY6K, CEA, and CYFRA 21-1 between tumorgroups and a healthy control group were analyzed by Mann-Whitney Utests. The levels of LY6K, CEA, and CYFRA 21-1 were further evaluated byreceiver-operating characteristic (ROC) curve analysis to determinecut-off levels with optimal diagnostic accuracy and likelihood ratios.The correlation coefficients between LY6K and CEA/CYFRA 21-1 werecalculated with Spearman rank correlation. Significance was defined asP<0.05.

Example 2

LY6K Expression in Lung and Esophageal Tumors, Cell Lines, and NormalTissues.

To search for novel molecules to serve as diagnostic biomarkers and/ortargets for development of therapeutic agents for lung and esophagealcancers, cDNA microarray analyses were applied to search for candidategenes that were transactivated in a large proportion of NSCLCs. Among27,648 genes screened, the LY6K transcript was identified as expressedspecifically in the great majority of the lung and esophageal cancersamples examined. Its transactivation was confirmed by semi-quantitativeRT-PCR experiments in 9 of 10 additional NSCLC tissues, in 8 of 8 ESCCtissues (FIGS. 1A and 1B).

Rabbit polyclonal antibody specific for human LY6K was subsequentlygenerated and used to confirm by western-blot analysis an expression ofLY6K protein in NSCLC samples in four representative pairs of NSCLCtissues and in four lung-cancer cell lines (two LY6K-positive and twoLY6K-negative cell lines) (FIG. 1C). The Immunofluorescence analysis wasperformed to examine the subcellular localization of endogenous LY6K inthe four lung-cancer cell lines (LC319, NCI-H1373, NCI-H226, and A427),and found that LY6K was located at cytoplasm of tumor cells withgranular appearance (FIG. 1D, left panels).

Since LY6K encodes GPI-anchored cell surface protein and some ofGPI-anchored proteins were known to be secreted into extra cellularspace (Nakatsura T, et al. Biochem Biophys Res Commun. 2003 Jun.20;306(1):16-25.), its presence in the culture media of the lung-cancercell lines was examined by ELISA. The amounts of detectable LY6K in theculture media was concordant to the expression levels of LY6K detectedwith semi-quantitative RT-PCR and western-blot analyses (FIG. 1D, rightpanel).

Northern blot analysis using an LY6K cDNA fragment as the probeidentified a transcript of about 1.8-kb that was highly and exclusivelyexpressed in testis among 23 normal human tissues examined (FIG. 2A).Expression of LY6K protein was subsequently examined in five normaltissues (heart, liver, lung, kidney, and testis) as well as lung cancersusing anti-LY6K antibody, and found that it was hardly detectable in theformer four tissues while positive LY6K staining appeared in testis andlung tumor tissues (FIG. 2B).

Example 3

Association of LY6K Over-Expression with Poor Clinical Outcomes amongNSCLC and ESCC Patients.

To verify the biological and clinicopathological significance of LY6K,the expression of LY6K protein was examined by means of tissuemicroarrays consisting of 413 NSCLC and 271 ESCC cases who underwentcurative surgical resection. LY6K staining was observed mainly in thecell membrane and cytoplasm of tumor cells, but was hardly detectable insurrounding normal tissues (FIGS. 3A and C).

A pattern of LY6K expression was classified on the tissue array rangingfrom absent/weak (scored as 0-1+) to strong (2+). Positive staining wasobserved in 224 (86.5%) of 259 lung ADC cases, 104 (92.0%) of 113 lungSCCs, 24 (85.7%) of 28 lung LCCs, and 13 (100%) of 13 lung ASCs, whileno staining was observed in any of the normal portions of the sametissues. Of the 413 NSCLC cases examined, LY6K was strongly stained in136 (32.9%; score 2+), weakly stained in 229 (55.5%; score 1+), and notstained in 48 cases (11.6%; score 0) (details are shown in Table 2A).NSCLC patients whose tumors showed strong LY6K expression revealedshorter tumor-specific survival compared to those with absent/weak LY6Kexpression (P=0.0026 by log-rank test; FIG. 3B).

Univariate analysis was also applied to evaluate associations betweenpatient prognosis and other factors including age (<65 versus 65>=),gender (female versus male), histological type (ADC versus non-ADC), pTclassification (T1, T2 vs T3, 4), pN classification (N0 versus N1, N2),and LY6K status (0, 1+versus 2+). Among those parameters, LY6K status(P=0.0028), elderly (P=0.0081), male (P=0.0022), non-ADC histologicalclassification (P=0.0090), advanced pT stage (P<0.0001), and advanced pNstage (P<0.0001) were significantly associated with poor prognosis(Table 2B). In multivariate analysis of the prognostic factors, strongLY6K expression, elderly, male gender, higher pT stage, and higher pNstage were indicated to be an independent prognostic factor (P=0.0201,<0.0001, 0.0166, 0.0002, and <0.0001, respectively; Table 2B).

Positive staining was observed in 257 (94.8%) of 271 esophageal cancer,while no staining was observed in any of the normal portions of the sametissues. LY6K was strongly stained in 176 (64.9%; score 2+), weaklystained in 81 (29.9%; score 1+), and not stained in 14 cases (5.2%;score 0) (details are shown in Table 3A). The median survival time ofESCC patients was significantly shorter in accordance with the higherexpression levels of LY6K (P=0.0455 by log-rank test; FIG. 3D).

Univariate analysis was also applied to evaluate associations betweenESCC patient prognosis and several factors including age (<65 versus65>=), gender (female versus male), pT stage (tumor depth; T1+T2 versusT3+T4), pN stage (node status; N0 versus N1), and LY6K status (score 0,1+versus 2+). Among those parameters, LY6K status (P=0.0467), male(P=0.031), advanced pT stage (P<0.0001), and advanced pN stage(P<0.0001) were significantly associated with poor prognosis (Table 3B).

In multivariate analysis, LY6K status did not reach the statisticallysignificant level as independent prognostic factor for surgicallytreated ESCC patients enrolled in this study (P=0.4479), while pT and pNstages as well as male gender did, suggesting the relevance of LY6Kexpression to these clinicopathological factors in esophageal cancer(P=0.0138, 0.0002, and <0.0001, respectively; Table 3B).

TABLE 2A Association between LY6K-positivity in NSCLC tissues andpatients' characteristics (n = 413) LY6K LY6K P-value strong weak LY6Kstrong Total positive positive absent vs weak/ n = 413 n = 136 n = 229 n= 48 absent Gender Male 284 102 150 32 NS Female 129 34 79 16 Age   <65202 72 109 21 NS (years) >=65 211 64 120 27 Histolog- ADC 259 72 152 35ical type SCC 113 48 56 9 0.0049⁺ Others 41 16 21 4 pT factor T1 + T2298 97 165 36 T3 + T4 115 39 64 12 NS pN factor N0 257 77 146 34 NS N1 +N2 156 59 83 14 ADC, adenocarcinoma; SCC, squamous-cell carcinomaOthers, large-cell carcinoma plus adenosquamous-cell carcinoma *ADCversus other histology ⁺P < 0.05 (Fisher's exact test) NS, nosignificance

TABLE 2B Cox's proportional hazards model analysis of prognostic factorsin patients with NSCLCs Hazards Variables ratio 95% CIUnfavorable/Favorable P-value Univariate analysis LY6K 1.545 1.161-2.056Strong(+)/Weak(+) 0.0028* or (−) Age (years) 1.471 1.105-1.956 65>=/<650.0081* Gender 1.664 1.201-2.306 Male/Female 0.0022* Histological 1.4581.099-1.934 others/ADC¹ 0.0090* type pT factor 1.987 1.480-2.667 T3 +T4/T1 + T2 <0.0001* pN factor 2.940 2.195-3.937 N1 + N2/N0 <0.0001*Multivariate analysis LY6K 1.414 1.056-1.893 Strong(+)/Weak(+) 0.0201*or (−) Age (years) 1.921 1.433-2.573 65>=/<65 <0.0001* Gender 1.5521.083-2.224 Male/Female 0.0166* Histological 1.226 0.926-1.731others/ADC¹ 0.1399 type pT factor 1.784 1.320-2.411 T3 + T4/T1 + T20.0002* pN factor 3.239 2.386-4.398 N1 + N2/N0 <0.0001* ¹ADC,adenocarcinoma *P < 0.05

TABLE 3A Association between LY6K-positivity in esophageal cancertissues and patients' characteristics (n = 271) LY6K LY6K P-value strongweak LY6K strong Total positive positive absent vs weak/ n = 271 n = 176n = 81 n = 14 absent Gender Male 245 160 74 11 NS Female 26 16 7 3 Age  <65 175 111 58 6 NS (years) >=65 96 65 23 8 pT factor T1 − T2 126 7343 10 0.0242* T3 + T4 145 103 38 4 pN factor N0 101 60 33 8 NS N1 + N2170 116 48 6 *P < 0.05 (Fisher's exact test) NS, no significance

TABLE 3B Cox's proportional hazards model analysis of prognostic factorsin patients with esophageal cancer Hazards Variables ratio 95% CIUnfavorable/Favorable P-value Univariate analysis LY6K 1.421 1.005-2.010Strong(+)/Weak(+) or (−) 0.0467* Age 1.023 0.734-1.426 65>=/<65 NS(years) Gender 3.145 1.472-6.720 Male/Female 0.031* pT factor 2.6861.905-3.786 T3 − T4/T1 − T2 <0.0001* pN factor 3.901 2.597-5.859 N1 +N2/N0 <0.0001* Multivariate analysis LY6K 1.234 0.871-1.749Strong(+)/Weak(+) or (−) N.S. Gender 2.605 1.216-5.582 Male/Female0.0138* pT factor 1.964 1.376-2.804 T3 − T4/T1 − T2 0.0002* pN factor3.004 1.970-4.580 N1 + N2/N0 <0.0001* *P < 0.05 NS, no significance

Example 4

Serum Levels of LY6K in Patients with NSCLC or ESCC.

Since the in vitro findings had suggested that LY6K could be secretedinto extra cellular space (FIG. 1D, right panel), it was examinedwhether LY6K was secreted into serum from patients with NSCLC or ESCC inorder to validate its potential as a novel serum biomarker. ELISAexperiments detected LY6K in serological samples from the great majorityof the 193 patients with lung or esophageal cancer.

The mean (+/−1SD) of serum LY6K in 112 lung cancer patients was331.3+/−739.3 pg/ml and those in 81 ESCC patients were 209.3+/−427.4pg/ml. In contrast, the mean (+/−1SD) serum levels of LY6K in 74 healthyindividuals were 34.2+/−65.3 pg/ml, and those in 65 patients with COPD,who were current and/or former smokers, were 54.4+/−233.8 pg/ml.

The levels of serum LY6K protein were significantly higher in lung oresophageal cancer patients than in healthy donors (between lung ADCpatients and healthy individuals, P<0.0001; between lung SCCs andhealthy individuals, P=0.0145; between ESCCs and healthy individuals,P<0.0001; Mann-Whitney U test), while the difference between healthyindividuals and COPD patients was not significant (P=0.5325; FIG. 4A).

According to histological types of lung cancer, the mean (+/−1SD) serumlevels of LY6K were 324.1+/−737.4 pg/ml in 85 ADC patients and those in27 SCC patients were 354.1+/−758.8 pg/ml; the differences between thetwo histologic types were not significant. High levels of serum LY6Kwere detected even in patients with earlier-stage tumors (FIG. 4B).Using receiver-operating characteristic (ROC) curves drawn with the dataof these 193 lung or esophageal cancer patients and 74 healthy donors(FIG. 5A and B), the cut-off level in this assay was set to provideoptimal diagnostic accuracy and likelihood ratios (minimal falsenegative and false positive results) for LY6K, i.e., 157.0 pg/ml with asensitivity of 33.2% (64/193) and a specificity of 4.1% (3/74).

According to tumor histology, the proportions of the serum LY6K-positivecases were 31.8% for ADC (27 of 85), 40.7% for SCC (11 of 27), and 32.1%for ESCC (26 of 81). The proportions of the serum LY6K-positive caseswere 9.2% (6/65) for COPD. ELISA experiments were then performed usingpaired preoperative and post-operative (2 months after the surgery)serum samples from lung cancer and ESCC patients to monitor the levelsof serum LY6K in the same patients. The concentration of serum LY6K wassignificantly reduced after surgical resection of primary tumors (FIG.8A). The serum LY6K values were further compared with the expressionlevels of LY6K in primary tumors in the same set of 16 NSCLC cases whoseserum had been collected before surgery (eight patients withLY6K-positive tumors and eight with LY6K-negative tumors). The levels ofserum LY6K showed good correlation with the expression levels of LY6K inprimary tumor (FIG. 8B). The results independently support the highspecificity and the great potentiality of serum LY6K as a biomarker fordetection of cancer at an early stage and for monitoring of the relapseof the disease.

Example 5

Comparison of LY6K with CEA and CYFRA 21-1 as Tumor Markers.

To evaluate the feasibility of using serum LY6K level as atumor-detection biomarker, serum levels of two conventional tumormarkers (CEA and CYFRA 21-1 for NSCLC patients) were also measured byELISA, using both in the same set of serum samples from cancer patientsand control individuals. ROC analyses determined the cut off value ofCEA for NSCLC detection to be 2.5 ng/ml (with a sensitivity of 39.8% anda specificity of 94.6%; FIG. 5A).

As shown in FIG. 5A, the correlation coefficient between serum LY6K andCEA values was not significant (Spearman rank correlation: rho=0.029,P=0.7583), indicating that measuring both markers in serum can improveoverall sensitivity for detection of NSCLC to 61.6%. False-positiveresults for either of the two tumor-markers among normal volunteers(control group) accounted for 9.5%, while the false-positive rates forCEA and LY6K in the same control group were 4.1% and 5.4%, respectively.According to tumor histology, the sensitivity of the combination ofserum LY6K and CEA as a tumor detection marker was 64.7% for ADC and51.6% for SCC, suggesting the usefulness of this combination for ADCdetection.

ROC analyses for the patients with NSCLC determined the cut-off value ofCYFRA 21-1 as 2.0 pg/ml, with a sensitivity of 39.8% and a specificityof 97.2% (FIG. 5B). The correlation coefficient between serum LY6K andCYFRA 21-1 values was not significant (Spearman rank correlation:rho=0.115, P=0.2165), also indicating that measurement of serum levelsof both markers can improve overall sensitivity for detection of NSCLCto 59.8%; for diagnosing NSCLC, the sensitivity of CYFRA 21-1 alone was39.8%. False-positive cases for either of the two tumor markers amongnormal volunteers (control group) were 6.8%, although the false-positiverates for CYFRA 21-1 in the same control group were 2.7%. According totumor histology, the sensitivity of the combination of serum LY6K andCYFRA 21-1 for the detection of tumors was 56.5% for ADC and 70.4% forSCC, indicating the usefulness of this combination for SCC detection.

As shown in FIG. 6A (left and middle panels), the correlationcoefficient between serum CEA and CYFRA 21-1 values was significant(Spearman rank correlation: rho=0.355, P=0.0002), whereas thecorrelation between serum LY6K and CEA values was not significant(Spearman rank correlation: rho=0.021, P=0.8275), indicating thatmeasuring both markers in serum can improve overall sensitivity fordetection of NSCLC to 61.6%. False-positive results for either of thetwo tumor-markers among normal volunteers (control group) accounted for9.5%, while the false-positive rates for CEA and LY6K in the samecontrol group were 4.1% and 5.4%, respectively. According to tumorhistology, the sensitivity of the combination of serum LY6K and CEA as atumor detection marker was 64.7% for ADC and 51.6% for SCC, suggestingthe usefulness of this combination for ADC detection.

The correlation coefficient between serum LY6K and CYFRA 21-1 values forNSCLC patients was not significant (Spearman rank correlation:rho=0.119, P=0.2114; Supplementary FIG. 6A, right panel), alsoindicating that measurement of serum levels of both markers can improveoverall sensitivity for detection of NSCLC to 59.8%; for diagnosingNSCLC, the sensitivity of CYFRA 21-1 alone was 33.9%. False-positivecases for either of the two tumor markers among normal volunteers(control group) were 6.8%, although the false-positive rates for CYFRA21-1 in the same control group were 2.7%. According to tumor histology,the sensitivity of the combination of serum LY6K and CYFRA 21-1 for thedetection of tumors was 56.5% for ADC and 70.4% for SCC, indicating theusefulness of this combination for SCC detection. Combination of LY6Kwith both CEA and CYFRA 21-1 indicated that 21 of 54 (38.9%) NSCLCpatients who were negative for both CEA and CYFRA 21-1, were diagnosedas LY6K-positive (FIG. 6B).

Serum levels of CEA and CYFRA 21-1 were further measured by ELISA in thesame set of serum samples from ESCC patients (FIG. 7A). The correlationcoefficient between serum LY6K and CEA values for ESCC patients was notsignificant (Spearman rank correlation: rho=0.153, P=0.0781; FIG. 7Amiddle panel), indicating that measuring both markers in serum canimprove overall sensitivity for detection of ESCC to 44.3%, whereas thesensitivity of CEA alone was 18.0%. The correlation between serum LY6Kand CYFRA 21-1 values for ESCC patients was also not significant(Spearman rank correlation: rho=0.034, P=0.6989; FIG. 7A, right panel).A combined assay for both LY6K and CYFRA 21-1 classified 52.5% of ESCCpatients as positive, while the sensitivity of CYFRA 21-1 alone was23.0%. Combination of LY6K with both CEA and CYFRA 21-1 indicated that16 of 40 (40.0%) ESCC patients who were negative for both CEA and CYFRA21-1, were diagnosed as LY6K-positive (FIG. 7B). The data clearlysuggest that serum LY6K levels were also high in certain proportion ofcancer patients that could not be diagnosed by the combination of CEAand CYFRA 21-1.

Example 6

Effect of LY6K-Small Interfering RNAs on Growth of NSCLC Cells andEsophageal Cancer Cells.

To assess whether LY6K plays a role in growth or survival of lung-cancercells, plasmids to express siRNA against LY6K (si-LY6K-1 and -2), alongwith two different control plasmids (siRNAs for EGFP and SCR) weredesigned, constructed, and transfected into lung cancer (RERF-LC-AI andLC319) and esophageal cancer (TE8) cells to suppress expression ofendogenous LY6K (representative data of RERF-LC-AI and TE8 was shown inFIG. 9). The amount of LY6K protein in the cells transfected withsi-LY6K-2 was significantly decreased in comparison with cellstransfected with any of the two control siRNAs or si-LY6K-1 (FIG. 9A andD). In accordance with its suppressive effect on protein levels of LY6K,transfected si-LY6K-2 caused significant decreases in colony numbers andcell viability measured by colony-formation (FIG. 9B) and MTT assays(FIG. 9C and E).

Discussions

As demonstrated herein, LY6K is expressed only in testis among thenormal tissues examined and is highly expressed in 88.2% of surgicallyresected samples from NSCLC patients and in 95.1% of those from ESCCpatients. The LY6K over-expression is associated with the shortercancer-specific survival period. Suppression of LY6K expression withsiRNA effectively suppresses growth of lung and esophageal cancer cellsthat expressed LY6K. These combined results strongly suggest that LY6Kis likely to be associated with highly malignant phenotype of thosetumors. Since LY6K is considered to be the cancer-testis antigens, LY6Kappears to be a good target for cancer immunotherapy.

It was also found that LY6K protein is secreted into serum from patientswith lung cancer or esophageal cancer that strongly expressed LY6K. Dueto the fact that concentration of serum LY6K was dramatically reducedafter surgical resection of primary tumors and the levels of serum LY6Kshowed good correlation with the expression levels of LY6K in primarytumor tissue in the same patients, positivity of serum LY6K appears tobe considerably correlated with the presence of primary tumors.Interestingly, the correlation coefficient between serum LY6K and CEA orCYFRA 21-1 values was not significant, whereas the correlationcoefficient between serum CEA and CYFRA 21-1 values was significant. Infact, 38.9-40.0% of NSCLC and ESCC patients who were negative for bothCEA and CYFRA 21-1, were diagnosed to be positive for LY6K (FIG. 6B and7B). An assay combining both LY6K and CEA/CYFRA 21-1 increased thesensitivity such that 64.7-70.4% of the patients with NSCLC and 52.5% ofESCC were diagnosed as positive, whereas 6.8-9.5% of healthy volunteerswere falsely diagnosed as positive. On the other hand, the sensitivityof the combination of conventional serum tumor marker, CEA and CYFRA21-1 in the same set of serum samples was 51.8% for NSCLC (53.0% for ADCand 48.1% for SCC) and 34.4% for ESCC, while false-positive cases foreither of the two tumor markers among normal volunteers (control group)were 6.8% (FIG. 6B and 7B). Although additional validation with a largerset of serum samples covering various clinical stages will be necessary,the data presented here sufficiently demonstrate a potential clinicalapplication of LY6K itself as a serologic/histochemical biomarker forlung and esophageal cancers. It should be also noted that activation ofLY6K was observed in more than half of a series of other types ofcancers such as cervical carcinomas (data not shown), suggesting itsdiagnostic and therapeutic application to a wide range of tumors.

INDUSTRIAL APPLICABILITY

The gene expression analysis of lung cancer and/or esophageal cancerdescribed herein, obtained through a combination of laser-capturedissection and genome-wide cDNA microarray, has identified LY6K genes astargets for cancer prevention and therapy. Based on the expression ofLY6K, the present invention provides molecular diagnostic markers foridentifying and detecting cancer, particularly lung cancer and/oresophageal cancer.

The methods described herein are also useful in the identification ofadditional molecular targets for prevention, diagnosis and treatment ofcancers such as lung cancer and/or esophageal cancer. The data reportedherein add to a comprehensive understanding of lung cancer and/oresophageal cancer, facilitate development of novel diagnosticstrategies, and provide clues for identification of molecular targetsfor therapeutic drugs and preventative agents. Such informationcontributes to a more profound understanding of lung and/or esophagealtumorigenesis, and provides indicators for developing novel strategiesfor diagnosis, treatment, and ultimately prevention of lung cancerand/or esophageal cancer.

Furthermore, the methods described herein are also useful in diagnosisof cancer, including lung and esophageal cancers, as well as theprognosis of the patients with these diseases. Moreover, the datareported here is also provide a likely candidate for development oftherapeutic approaches for cancer including lung and esophageal cancers.

All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety.However, nothing herein should be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it is to be understood thatthe foregoing description is exemplary and explanatory in nature and isintended to illustrate the invention and its preferred embodiments.Through routine experimentation, one skilled in the art will readilyrecognize that various changes and modifications can be made thereinwithout departing from the spirit and scope of the invention. Furtheradvantages and features will become apparent from the claims filedhereafter, with the scope of such claims to be determined by theirreasonable equivalents, as would be understood by those skilled in theart. Thus, the invention is intended to be defined not by the abovedescription, but by the following claims and their equivalents.

1. A method of diagnosing esophageal cancer or a predisposition fordeveloping esophageal cancer in a subject, including the step ofdetermining a level of expression of LY6K in a biological sample from apatient, wherein an increase in said sample expression level as comparedto a normal control expression level of said gene indicates that saidsubject suffers from or is at risk of developing esophageal cancer. 2.The method of claim 1, wherein said sample expression level is at least10% greater than said normal control level.
 3. The method of claim 1,wherein said esophageal cancer is esophageal squamous-cell carcinoma. 4.The method of claim 1, wherein said biological sample comprises anepithelial cell.
 5. The method of claim 1, wherein said biologicalsample comprises an esophageal cancer cell.
 6. The method of claim 1,wherein said biological sample comprises an epithelial cell from anesophageal cancer.
 7. The method of claim 1, wherein gene expressionlevel is determined by a method selected from the group consisting of:(a) detecting mRNA of LY6K, (b) detecting a protein encoded by LY6K, and(c) detecting a biological activity of a protein encoded by LY6K.
 8. Amethod of screening for a compound for treating or preventing lungcancer or esophageal cancer, said method comprising the steps of: (a)contacting a test compound with a polypeptide encoded by LY6K; (b)detecting the binding activity between the polypeptide and the testcompound; and (c) selecting the test compound that binds to thepolypeptide.
 9. A method of screening for a compound for treating orpreventing lung cancer or esophageal cancer, said method comprising thesteps of: (a) contacting a candidate compound with a cell expressingLY6K; and (b) selecting the candidate compound that reduces theexpression level of LY6K, as compared to an expression level detected inthe absence of the candidate compound.
 10. The method of claim 9,wherein said cell comprises a lung cancer cell or an esophageal cancercell.
 11. A method of screening for a compound for treating orpreventing lung cancer or esophageal cancer, said method comprising thesteps of: (a) contacting a test compound with a polypeptide encoded byLY6K; (b) detecting the biological activity of the polypeptide of step(a); and (c) selecting the test compound that suppresses the biologicalactivity of the polypeptide encoded by LY6K, as compared to thebiological activity of said polypeptide detected in the absence of thetest compound.
 12. The method of claim 11, wherein the biologicalactivity of the polypeptide is cell proliferative activity.
 13. A methodof screening for compound for treating or preventing lung cancer oresophageal cancer, said method comprising the steps of: (a) contacting acandidate compound with a cell into which a vector, comprising thetranscriptional regulatory region of LY6K and a reporter gene that isexpressed under the control of the transcriptional regulatory region,has been introduced; (b) measuring the expression level or activity ofsaid reporter gene; and (c) selecting the candidate compound thatreduces the expression level or activity of said reporter gene, ascompared to an expression level or activity detected in the absence ofthe candidate compound.
 14. A method of treating or preventingesophageal cancer in a subject comprising administering to said subjectan antisense composition, said antisense composition comprising anucleotide sequence complementary to a coding sequence of LY6K.
 15. Amethod for treating or preventing esophageal cancer in a subjectcomprising the step of administering to said subject a pharmaceuticallyeffective amount of an antibody, or an immunologically active fragmentthereof, that binds to a protein encoded by LY6K.
 16. A composition fortreating or preventing esophageal cancer, said composition comprising apharmaceutically effective amount of an antisense polynucleotide againstLY6K.
 17. A composition for treating or preventing esophageal cancer,said composition comprising a pharmaceutically effective amount of anantibody or an immunologically active fragment thereof that binds to aprotein encoded by LY6K.
 18. A method for diagnosing cancer in asubject, comprising the steps of: (a) collecting a blood sample from asubject to be diagnosed; (b) determining a level of LY6K in the bloodsample; (c) comparing the LY6K level determined in step (b) with that ofa normal control; and (d) judging that a high LY6K level in the bloodsample, as compared to the normal control, indicates that the subjectsuffers from cancer.
 19. The method of claim 18, wherein the cancer isesophageal and/or lung cancer.
 20. The method of claim 18, wherein theblood sample is selected from the group consisting of whole blood,serum, and plasma.
 21. The method of claim 18, wherein the LY6K level isdetermined by detecting the LY6K protein in the serum.
 22. The method ofclaim 21, wherein the LY6K protein is detected by immunoassay.
 23. Themethod of claim 22, wherein the immunoassay comprises a step for bindingthe LY6K protein with an antibody which binds the LY6K protein at theamino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19 of the protein.24. The method of claim 22, wherein the immunoassay is an ELISA.
 25. Themethod of claim 24, wherein the ELISA is sandwich method.
 26. The methodof claim 18, wherein said method further comprises: (e) determining thelevel of one or more other cancer-associated proteins in the bloodsample; (f) comparing the protein level(s) determined in step (e) withthat of a normal control; and (g) judging that high level(s) of othercancer-associated proteins in the blood sample when compared to thenormal control indicates that the subject suffers from cancer.
 27. Themethod of claim 26, wherein said the cancer-associated protein is eitheror both of CEA and CYFRA 21-1.
 28. A kit for detecting a cancer, whereinthe kit comprises: (a) an immunoassay reagent for determining a level ofLY6K in a blood sample; and (b) a positive control sample for LY6K. 29.The kit of claim 28, wherein the cancer is esophageal and/or lungcancer.
 30. The kit of claim 29, wherein the positive control sample ispositive for LY6K.
 31. The kit of claim 30, wherein the positive controlsample is liquid form.
 32. The kit of claim 31, wherein the positivecontrol sample is blood sample which comprises a higher than normallevel of LY6K.
 33. The kit of claim 28, wherein the immunoassay reagentis antibody which recognizes amino acid sequence comprising SEQ ID NO:18 or
 19. 34. The kit of claim 28, which further comprises: (c) animmunoassay reagent for determining the level of one or more othercancer-associated proteins in a blood sample; and (d) a positive controlfor the other cancer-associated proteins.
 35. The kit of claim 34,wherein said cancer-associated protein is either or both of CEA andCYFRA 21-1.
 36. A method for assessing the prognosis of a patient withcancer, which method comprises the steps of: (a) detecting theexpression level of an LY6K gene in a patient-derived biological sample;(b) comparing the detected expression level to a control level; and (c)determining the prognosis of the patient based on the comparison of (b).37. The method of claim 36, wherein the control level is a goodprognosis control level and an increase of the expression level ascompared to the control level is determined as poor prognosis.
 38. Themethod of claim 37, wherein the increase is at least 10% greater thansaid control level.
 39. The method of claim 36, wherein said expressionlevel is determined by any one method selected from the group consistingof: (a) detecting mRNA of the LY6K gene; (b) detecting the LY6K protein;and (c) detecting the biological activity of the LY6K protein.
 40. Themethod of claim 36, wherein said expression level is determined bydetecting hybridization of a probe to a gene transcript of the LY6Kgene.
 41. The method of claim 40, wherein the hybridization step iscarried out on a DNA array.
 42. The method of claim 36, wherein saidexpression level is determined by correlating the binding of an antibodyagainst the LY6K protein with the expression level of the LY6K gene. 43.The method of claim 42, wherein the antibody recognizes amino acidsequence comprising SEQ ID NO: 18 or
 19. 44. The method of claim 36,wherein said biological sample comprises sputum or blood.
 45. The methodof claim 36, wherein the cancer is esophageal and/or lung cancer.
 46. Akit for assessing the prognosis of a patient with cancer, whichcomprises a reagent selected from the group consisting of: (a) a reagentfor detecting mRNA of an LY6K gene; (b) a reagent for detecting an LY6Kprotein; and (c) a reagent for detecting the biological activity of anLY6K protein.
 47. The kit of claim 46, wherein the reagent is anantibody against the LY6K protein.
 48. The kit of claim 47, wherein theantibody recognizes amino acid sequence comprising SEQ ID NO: 18 or 19.49. The kit of claim 46, wherein the cancer is esophageal and/or lungcancer. 50.-66. (canceled)